Manufacturing method of a cell structure

ABSTRACT

The present disclosure aims to provide a manufacturing method of a cell structure. The manufacturing method comprises producing a coated region in which a culturing surface is coated with a temperature-responsive polymer or a temperature-responsive polymer composition, forming a droplet of a cell suspension in the coated region, and performing cell culturing in the droplet. A surface zeta potential of the coated region is 0 mV to 50 mV.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation application of U.S. patentapplication Ser. No. 16/073,879 filed Jul. 30, 2018, which is a NationalStage Application of PCT/JP2017/003879 filed Jan. 27, 2017, which claimspriorities based on Japanese Patent Application No. 2016-016459 filedJan. 29, 2016, Japanese Patent Application No. 2016-124458 filed Jun.23, 2016, Japanese Patent Application No. 2016-162950 filed Aug. 23,2016, Japanese Patent Application No. 2016-148977 filed Jul. 28, 2016,Japanese Patent Application No. 2016-026293 filed Feb. 15, 2016,Japanese Patent Application No. 2016-053081 filed Mar. 16, 2016,Japanese Patent Application No. 2016-053082 filed Mar. 16, 2016,Japanese Patent Application No. 2016-053084 filed Mar. 16, 2016,Japanese Patent Application No. 2016-053086 filed Mar. 16, 2016. Thedisclosures of the prior applications are hereby incorporated byreference herein in their entirety.

TECHNICAL FIELD

The present disclosure relates to a method of efficiently manufacturinga cell mass, a cell structure, or a three-dimensional tissue body usinga culturing surface coated with a temperature-responsive polymer or atemperature-responsive polymer composition, and to a cell mass, a cellstructure, or a three-dimensional tissue body obtainable using thismethod.

In particular, Aspect (I) of the present disclosure relates to amanufacturing method of a chondrocyte mass and a graft material, and toa chondrocyte mass, a graft material, and a composite material. Aspect(II) of the present disclosure relates to a culture method of epithelialcells, a manufacturing method of a cell structure, and a cell culturecontainer for epithelial cells. Aspect (III) of the present disclosurerelates to a production apparatus of a three-dimensional tissue body anda production method of a three-dimensional tissue body. Aspect (IV) ofthe present disclosure relates to a manufacturing method of a cellstructure. Aspect (V) of the present disclosure relates to amanufacturing method of a cell structure, a cell structure, and a cellculture container. Aspect (VI) of the present disclosure relates to amanufacturing method of a cell structure. Aspect (VII) of the presentdisclosure relates to a manufacturing method of a cell structure.

BACKGROUND

With regard to Aspect (I), demand has increased for customized medicaltreatment to improve the quality of life (QOL) of patients. Regenerativemedicine, which aims to regenerate tissues and organs with impaired orlost functions using a patient's own cells, plays a leading role incustomized medical treatment.

Regenerative medicine requires operations to culture cells collectedfrom a patient's tissue in a cell culture container, to form tissue, andthen to graft the tissue onto the patient. For this reason, a techniquefor culturing cells to form a cell structure such as tissue and atechnique for collecting the cell structure without altering its stateare desired.

In general, cells extracted from a living organism suffer variousstresses that disturb gene regulation, often provokingdedifferentiation. Dedifferentiation is also often necessary to growcells. Consequently, the initial gene expression state of the cellsoften cannot be maintained by culturing cells collected from a patientunder simple culture conditions. This prevents formation of cellstructures, and therefore of tissue. Furthermore, advanced functions ofthe cells cannot be achieved. For example, when cells are cultured in atypical polystyrene cell culture dish, the cells merely form a singlelayer structure, making it difficult to form a cell structure similar tothe structure seen in highly differentiated cells, such as the pelletstructure adopted by chondrocytes in a living organism. Many specificfunctions of chondrocytes are also lost.

To address these problems, cell culture methods to construct athree-dimensional structure that imitates the structure of tissue havebeen developed, such as methods for a spheroid culture, cluster culture,pellet culture, three-dimensional carrier culture, and the like. A cellculture method of producing cells that have a three-dimensionalstructure by using an extracellular matrix with a three-dimensionalstructure as a cell culture scaffold is known.

In the field of biological tissue regeneration, related techniques arebeing perfected, such as an unattended, automated cell culture method,drug discovery for differentiation control, and methods of testing forvirus infection. In light of this, the free design of high-orderphysical structures that imitate the structures in living organisms hasbecome an active area of research (see Non-patent Literature (NPL) 1,2).

With regard to three-dimensional culturing of cartilage, an example ofsuccessfully producing a cartilage disk measuring 10 mm in diameter and1 mm thick by injecting dedifferentiated chondrocytes into a shaped moldand inducing differentiation by chemically stimulating the cells withBMP2, b-FGF, or the like has been reported in particular.

With regard to Aspect (II), epithelial cells have weak adhesiveness tocell culture containers, which has made it extremely difficult toculture epithelial cells using a regular cell culture container. Cellculture containers that improve the adhesiveness of cells by beingcoated with a cell adhesion factor, such as a collagen-coated cellculture container (see Patent Literature (PTL) 1), a fibronectin-coatedcell culture container (see PTL 2), a laminin-coated cell culturecontainer (see PTL 3), and the like are known. A method using a celladhesion factor that is a chemical synthetic substance, however, isdesired to conserve animal resources and to avoid the unknown substancesor pathogenic substances that might be included when using a celladhesion factor derived from natural products.

Furthermore, the adhesiveness between the epithelial cells and theculturing surface is insufficient with a method using a known celladhesion factor, such as collagen, fibronectin, laminin, or the like. Acell culture container with excellent adhesiveness of epithelial cellsthus is currently in demand.

With regard to Aspect (III), a hanging drop method (see NPL 3), alow-adhesion U-shaped bottom culture dish (see PTL 4), and the like forsimple cell structures such as spheres and sheets are known asproduction methods of cell structures and synthetic tissue bodies with athree-dimensional structure.

Cell structures formed using a 3D printer are also known as cellstructures with a complex three-dimensional shape.

With regard to Aspect (IV), important experimental techniques in thefield of biology include cell culture techniques developed around theyear 1900. Initial development of these techniques focused only onconditioning cells, such as optimizing the medium components, andtechniques for single layer cultures and suspension cultures were mainlystudied.

In recent years, it has become clear that various properties, stimulusresponsiveness, cell functions, and the like differ between single layercultured cells and cells in living tissue. Instead of single layercultures that form a single layer structure, demand is increasing for 3Dcultures, in particular spheroid cultures, that form a 3D structureresembling the tissue structure in a living organism (see NPL 4).

Traditional techniques that have been actively developed include atechnique for embedding cells in a 3D gel formed by suspending cells ina protein solution and causing the cell suspension to gel in reaction toa certain trigger (heat, light, a chemical crosslinking agent, or thelike), a technique for grafting cells onto a porous scaffold, and atechnique for producing a laminate of a cell sheet using a culture dishwith hardened NIPAM.

Techniques developed in recent years include a technique of causingcells to precipitate on a non-adhesive round bottom, as withPrimeSurface by Sumitomo Bakelite Co. or the like; a technique ofheightening the migration property of cells adhered to a culturingsurface by providing a smooth surface with a regular pattern ofunevenness by laser processing, thereby inducing self-assembly of cellson the culturing surface, as with Nano Culture Plate by JSR Co., NanoPillar Plate by Hitachi, or the like; and a technique of using a culturedish with countless holes approximately 100 μm to 500 μm in diameter and500 μm deep to precipitate cells seeded in each hole onto the bottom ofthe hole, as with Elplasia by Kuraray Co., EZSPHERE by Iwaki & Co., orthe like.

A hanging drop method to manufacture a spheroidal cell structure in adroplet by producing a droplet of a cell suspension at the tip of atubular member and holding the droplet for a predetermined time period(such as approximately 2 weeks) while maintaining the spherical shape ofthe droplet using the surface tension of the droplet has also beendeveloped recently.

With regard to Aspect (V), demand has increased for customizedhealthcare to improve the QOL of patients. Regenerative medicine, whichaims to regenerate tissues and organs with impaired or lost functionsusing a patient's own cells, plays a leading role in customized medicaltreatment.

Regenerative medicine requires operations to culture cells collectedfrom a patient's tissue in a cell culture container, to form tissue, andthen to transplant the tissue into the patient. For this reason, atechnique for culturing cells to form a cell structure such as tissueand a technique for collecting the cell structure without altering itsstate are desired.

In general, cells extracted from a living organism suffer variousstresses that disturb gene regulation, often provokingdedifferentiation. Dedifferentiation is also often necessary to growcells. Consequently, the initial gene expression state of the cellsoften cannot be maintained by culturing cells collected from a patientunder simple culture conditions. This prevents formation of cellstructures, and therefore of tissue. Furthermore, advanced functions ofthe cells cannot be achieved. For example, when cells are cultured in atypical polystyrene cell culture dish, the cells merely form a singlelayer structure, making it difficult to form a cell structure similar tothe structure seen in highly differentiated cells, such as the pelletstructure adopted by chondrocytes in a living organism. Many specificfunctions of chondrocytes are also lost.

To address these problems, cell culture methods to construct athree-dimensional structure that imitates the structure of tissue havebeen developed, such as methods for a spheroid culture, cluster culture,pellet culture, three-dimensional carrier culture, and the like. A cellculture method of producing cell structures that have athree-dimensional structure by using an extracellular matrix with athree-dimensional structure as a cell culture scaffold is known (see PTL5).

Other techniques that have been developed to produce three-dimensionalcell structures include a technique of using a low-adhesion culture dishwith a U-shaped bottom and a hanging drop method.

In recent years, a method of easily manufacturing three-dimensional cellstructures by seeding and culturing cells on a culturing surface coatedwith a special temperature-responsive polymer and/ortemperature-responsive polymer composition has been reported (see PTL6).

With regard to Aspect (VI), research on cardiac dysfunction has led to aheart disease model that places an animal's biological heart in a statesuch as cardiac failure. A widely known example is a model animalproduced with a method such as provoking autoimmune myocarditis byoccluding coronary arteries, administering drugs, or injecting cardiacmyosin intramuscularly into a lower extremity.

The heart is an extremely important organ for maintaining life and alsoaffects the state of other organs, making production of a reproducibleheart disease model with reduced cardiac function difficult. In the caseof coronary artery occlusion, for example, a minor occlusion yieldslittle difference from a healthy state, whereas the animal is lostquickly with an even slightly significant occlusion. The differencebetween these two extremes is small, making adjustment extremelydifficult. In a model of myocarditis due to an autoimmune reaction uponadministration of myosin, the medical state of cardiac tissue differsgreatly depending on the degree of the immune reaction. Moreover, thecardiac function itself is greatly affected by the state of other organsseparate from the heart. Hence, it is extremely difficult to performstable experiments or construct a reproducible experiment system.

When a produced heart disease model organ is extracted from a livingorganism, it is also difficult to maintain the cardiomyocytes, whichrequire much oxygen.

The use of laboratory animals also raises issues regarding animalwelfare and ethics.

Typically, in tissue that has suffered cardiac failure or the like, itis known that cardiomyocytes necrotize due to partial obstruction oftubular arteries, viral or bacterial infection, an autoimmune reaction,or the like, and that the necrotic cardiomyocytes are replaced byexcessive growth of fibroblasts.

Attempts are thus being made to reproduce heart disease tissue that hassuffered cardiac failure or the like in a test tube. To do so, it isnecessary to form a cell structure by coculturing cardiomyocytes withrapid-growing fibroblasts while protecting the cardiomyocytes, which aresusceptible to hypoxia. This is difficult with a known hanging dropmethod or low-adhesion culture dish, which require 1 to 2 weeks toproduce cell structures.

A known method of manufacturing a cell mass efficiently and rapidly usesa cell culture container coated with a particular temperature-responsivepolymer (see PTL 6).

With regard to Aspect (VII), a hepatic failure model animal is used inresearch on liver regeneration therapy, a representative example ofwhich is the transplanting of Muse cells (pluripotent stem cells). Knownproduction methods of hepatic failure model animals include, forexample, methods for partial excision of the liver, printing of portalveins and upstream blood vessels, and repeated administration ofliver-damaging drugs such as carbon tetrachloride. In many cases, adiseased liver is used in an in vivo experiment, without being extractedfrom the animal's body.

Experiments using animals raise issues regarding animal welfare andethics, and the balance between the purpose of the experiment, thesuccess rate, and the value of the obtained findings is important.

In methods for partial excision of the liver, the degree of hepaticfailure and the individual animal's state greatly depend on theoperator's technique. In methods using liver-damaging drugs, well-knownstandard protocols exist, but repeated administration is oftennecessary, and it is difficult to control the degree of progress ofsymptoms. It is thus difficult to stably construct a reproducibleexperiment system using a hepatic failure model animal.

Hepatocytes are actively being cultured, but fibroblasts or the likemust be cocultured to reproduce a hepatic failure model. However, theadhesiveness to the culture dish and the culture method differ betweenhepatocytes, which are epithelial cells, and fibroblasts, which aremesenchymal cells, making it difficult to produce a three-dimensionalcell structure that includes hepatocytes and fibroblasts with a knownhanging drop method or low-adhesion culture dish, which require 1 to 2weeks for production.

A known method of manufacturing a cell mass efficiently and rapidly usesa cell culture container coated with a particular temperature-responsivepolymer (see PTL 6).

CITATION LIST Patent Literature

-   PTL 1: JPH05260950A-   PTL 2: JPH06014764A-   PTL 3: JPH08173144-   PTL 4: JP2009050194-   PTL 5: JP2010524458-   PTL 6: JP5746240B2

Non-Patent Literature

-   NPL 1: M. Matsusaki et al, Adv. Healthcare Mater., 2, 534 (2013)-   NPL 2: M. Matsusaki et al, Biochem. Biophys. Res. Commun., 457, 363    (2015)-   NPL 3: Keller G. M. et al., Curr. Opin. Cell Biol., 7, 862-869    (1995)-   NPL 4: Nature, Vol 424, P870-872, 21 Aug. 2003.

SUMMARY Technical Problem

The present disclosure aims to provide a method of efficientlymanufacturing a cell mass, a cell structure, or a three-dimensionaltissue body using a culturing surface coated with atemperature-responsive polymer or a temperature-responsive polymercomposition.

With regard to Aspect (I), it has been reported that use of theaforementioned known methods to produce cultured cartilage with a largersize and/or a complex structure leads to necrosis of cells inside thestructure, causing death.

Therefore, Aspect (I) aims to easily manufacture a chondrocyte mass anda graft material, along with a composite material, that are useful fortreatment of joints, the trachea, the nose, and the like.

Aspect (II) aims to provide a culture method of epithelial cells thattend not to adhere to a cell culture container, a manufacturing methodof a cell structure that includes epithelial cells that tend not toadhere to a cell culture container, and a cell culture container thatallows culturing of epithelial cells and manufacturing of a cellstructure thereof.

With regard to Aspect (III), the provision of oxygen and nutrients tocells inside the cell structure depends on the concentration gradientdiffusion in a hanging drop method or a method using a low-adhesionU-shaped bottom culture dish or the like. Hence, the size is restricted,with a diameter of approximately 0.1 mm typically being considered themaximum. The shape is also limited to being spherical.

A production method of a cell structure using a 3D printer uses a cellsuspension, in which cells are individually dispersed using an enzymesuch as trypsin, and ejects the cells from a nozzle to produce a cellstructure. With this method, an adhesion factor or the like needs to beejected simultaneously from the outside to the area surrounding theindividually ejected cells for the ejected cells to cohere. Thisadhesion factor was not secreted by the cells, however, and theresulting three-dimensional cell structure does not have sufficientcohesion between cells or sufficient cell activity.

In recent years, the importance of techniques for culturing cells in acell culture container to form a three-dimensional cell structureimitating a tissue structure, such as a ringed shape or a luminal shape,has increased from the perspective of regenerative medicine, which aimsto regenerate tissues and organs with impaired or lost functions. Apartfrom cell structures with cells as the principal component, a method isalso now in demand for producing a cell structure with a principalcomponent of extracellular matrix as a three-dimensional cell structureimitating a tissue structure.

No method, however, for easily forming a three-dimensional tissue bodythat imitates a tissue structure with a ringed shape, a luminal shape,or the like is currently known.

Accordingly, Aspect (III) aims to provide a production apparatus of athree-dimensional tissue body that can easily yield a three-dimensionaltissue body with a ringed shape, a luminal shape, or the like, and toprovide a production method of a three-dimensional tissue body that caneasily yield a three-dimensional tissue body with a ringed shape, aluminal shape, or the like.

With respect to Aspect (IV), manufacturing of spheroidal cell structuresusing the aforementioned known methods has the problem of low vitalityof the cell structures, a difficulty in achieving the desired size, andtrouble adjusting the shape. In particular, if the size and shape of thecell structures are not uniform, it becomes necessary to sort themanufactured cell structures, complicating the manufacturing process andincreasing costs.

Therefore, Aspect (IV) aims to easily manufacture cell structures havinga desired size and a well-defined spheroidal shape.

With respect to Aspect (V), the aforementioned spheroid culture methodand the like have the problems of a small diameter of approximately 10μm and the ability only to produce spheroids (aggregates of multiplecells) with a weak intercellular network.

The aforementioned hanging drop method and technique using alow-adhesion culture dish with a U-shaped bottom can only yieldspheroids that are substantially a true sphere and cannot yieldspheroids with a cell-specific form, such as a cobblestone or spindleform.

The cell structures obtainable with a method using the aforementionedspecial polymer and/or polymer composition developed in recent years donot always reach the desired form, and the conditions for this methodhave room for optimization and improvement.

Therefore, Aspect (V) aims to manufacture a cell structure with thedesired form by controlling the aggregation mode of cells.

With regard to Aspect (VI), the method disclosed in PTL 6 does notconsider reproduction of heart disease tissue that has suffered cardiacfailure or the like. A method of reproducing heart disease in a testtube is currently in demand.

Accordingly, Aspect (VI) aims to provide a manufacturing method of acell structure for easy formation of a cell structure that includescardiomyocytes and fibroblasts and is useful as a heart disease model.

With regard to Aspect (VII), the method disclosed in PTL 6 does notconsider reproduction of hepatic failure tissue. A method of reproducinghepatic failure in a test tube is currently in demand.

Accordingly, Aspect (VII) aims to provide a manufacturing method of acell structure for easy formation of a cell structure that includescardiomyocytes and fibroblasts and is useful as a hepatic failure model.

Solution to Problem

The following is a summary of Aspects (I) to (VII) of the presentdisclosure.

A manufacturing method of a chondrocyte mass of Aspect (I) includes aseeding and culturing step of seeding, in the presence of a cell mass,cells capable of differentiating into chondrocytes onto a coatedculturing surface coated with a temperature-responsive polymer or atemperature-responsive polymer composition and culturing the cell massand the cells capable of differentiating into chondrocytes to produce achondrocyte mass.

In the manufacturing method of a chondrocyte mass of Aspect (I), thecell mass is preferably produced by seeding and culturing the cellscapable of differentiating into chondrocytes.

In the manufacturing method of a chondrocyte mass of Aspect (I), theseeding and culturing step is preferably performed a plurality of times.

In the manufacturing method of a chondrocyte mass of Aspect (I), thecoated culturing surface is preferably surrounded by a cell non-adhesivewall.

In the manufacturing method of a chondrocyte mass of Aspect (I), thecoated culturing surface preferably has a width of 3 mm or less, and thewall preferably has a height of 3 mm or less.

In the manufacturing method of a chondrocyte mass of Aspect (I), anamount of the temperature-responsive polymer and thetemperature-responsive polymer composition that the coated culturingsurface has per unit area is preferably 0.1 μg/cm² to 3.0 μg/cm².

In the manufacturing method of a chondrocyte mass of Aspect (I), thecells capable of differentiating into chondrocytes are preferably seededat a cell density of 0.3×10⁴ cells/cm² to 10.0×10⁵ cells/cm² in theseeding and culturing step.

A chondrocyte mass of Aspect (I) is manufactured using theabove-described manufacturing method of a chondrocyte mass of thepresent disclosure.

The chondrocyte mass of Aspect (I) preferably has a donut shape.

A manufacturing method of a graft material of Aspect (I) includesseeding mesenchymal cells in the presence of the chondrocyte mass of thepresent disclosure and culturing the chondrocyte mass and themesenchymal cells to produce a graft material.

A graft material of Aspect (I) is manufactured using the manufacturingmethod of a graft material of the present disclosure.

A composite material of Aspect (I) includes the chondrocyte mass of thepresent disclosure on an outer surface of a tubular structure.

The composite material of Aspect (I) preferably further includes a corematerial inside the tubular structure.

Aspect (II) provides a culture method of epithelial cells, the culturemethod including a production step of producing a temperature-responsivepolymer or a temperature-responsive polymer composition, a culturecontainer preparation step of forming a coated region A by coating atleast a portion of a culturing surface of a cell culture container withthe temperature-responsive polymer or the temperature-responsive polymercomposition to prepare a coated cell culture container including thecoated region A, a seeding step of seeding epithelial cells in thecoated cell culture container, and a culturing step of culturing theepithelial cells adhered to the coated region A. The concentration ofthe temperature-responsive polymer or the temperature-responsive polymercomposition in the coated region A is 0.3 pg/mm² or more.

In the culture method of epithelial cells of Aspect (II), at least aportion of the culturing surface of the cell culture containerpreferably includes a depression located within the coated region A.

Aspect (II) also provides a manufacturing method of a cell structure,the manufacturing method including a production step of producing atemperature-responsive polymer or a temperature-responsive polymercomposition, a culture container preparation step of forming a coatedregion A by coating at least a portion of a culturing surface of a cellculture container with the temperature-responsive polymer or thetemperature-responsive polymer composition to prepare a coated cellculture container including the coated region A, a seeding step ofseeding epithelial cells in the coated cell culture container, and aculturing step of forming an aggregated cell structure from theepithelial cells to obtain a cell structure adhered to the coated regionA. The concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition in the coated region A is 0.3pg/mm² or more.

In the manufacturing method of a cell structure of Embodiment (II), inthe culture container preparation step, a coated region B coated withthe temperature-responsive polymer or the temperature-responsive polymercomposition is preferably formed on at least a portion of the culturingsurface of the cell culture container at a different position than thecoated region A, and the concentration of the temperature-responsivepolymer or the temperature-responsive polymer composition in the coatedregion B is preferably less than 200 pg/mm².

In the manufacturing method of a cell structure of Embodiment (II), atleast a portion of the culturing surface of the cell culture containerpreferably includes a depression located within the coated region A.

Aspect (II) also provides a cell culture container for epithelial cells,the cell culture container including a coated region A, coated with atemperature-responsive polymer or a temperature-responsive polymercomposition, on at least a portion of a culturing surface. Theconcentration of the temperature-responsive polymer or thetemperature-responsive polymer composition in the coated region A is 0.3pg/mm² or more.

The cell culture container for epithelial cells of Embodiment (II)preferably further includes a coated region B, coated with atemperature-responsive polymer or a temperature-responsive polymercomposition, on at least a portion of the culturing surface at adifferent position than the coated region A, and the concentration ofthe temperature-responsive polymer or the temperature-responsive polymercomposition in the coated region B is preferably less than 200 pg/mm².

In the cell culture container for epithelial cells of Embodiment (II),at least a portion of the culturing surface of the cell culturecontainer preferably includes a depression located within the coatedregion A.

Aspect (III) provides a production apparatus of a three-dimensionaltissue body, the production apparatus including a culturing surfacehaving one or more through holes, a shaft inserted through the one ormore through holes, and one or more coated culturing surfaces where theculturing surface is coated with a temperature-responsive polymer or atemperature-responsive polymer composition. At least one of the one ormore through holes is located within one of the one or more coatedculturing surfaces, and the culturing surface is movable in an extendingdirection of the shaft.

The production apparatus of a three-dimensional tissue body preferablyincludes a plurality of the culturing surfaces, and the shaft ispreferably inserted through the through holes of the plurality of theculturing surfaces.

Aspect (III) also provides a production method of a three-dimensionaltissue body using the production apparatus of a three-dimensional tissuebody, the production method including a seeding step of seeding at leastone type of cells on the coated culturing surface, and a culturing stepof culturing the seeded cells to obtain a ringed three-dimensionaltissue body wound around the shaft.

The production method of a three-dimensional tissue body preferablyfurther includes repetition of a culturing surface moving step of movingthe culturing surface in the extending direction of the shaft afterobtaining the ringed three-dimensional tissue body wound around theshaft, a seeding step of seeding at least one type of cells on thecoated culturing surface after the culturing surface is moved, and aculturing step of culturing the seeded cells to obtain another ringedthree-dimensional tissue body wound around the shaft adjacent to theringed three-dimensional tissue body wound around the shaft.

In the production method of a three-dimensional tissue body, the cellsare preferably seeded on all of the coated culturing surfaces, and theseeded cells are preferably cultured to obtain a three-dimensionaltissue body.

The production method of a three-dimensional tissue body preferablyincludes obtaining a three-dimensional tissue body including the cellsseeded in the seeding step.

The production method of a three-dimensional tissue body preferablyincludes removing the cells after the culturing step, and obtaining athree-dimensional tissue body including a substance secreted by thecells.

In the production method of a three-dimensional tissue body, thesubstance is preferably a protein.

A manufacturing method of a cell structure of Aspect (IV) includesproducing a coated region in which a culturing surface is coated with atemperature-responsive polymer or a temperature-responsive polymercomposition, forming a droplet of a cell suspension in the coatedregion, and performing cell culturing in the droplet. The surface zetapotential of the coated region is 0 mV to 50 mV.

In the manufacturing method of a cell structure of Aspect (IV), thecontact angle of water relative to the coated region is preferably 50°to 90°.

The manufacturing method of a cell structure of Aspect (IV) preferablyincludes producing a plurality of the coated regions on the culturingsurface.

The manufacturing method of a cell structure of Aspect (IV) preferablyincludes forming a plurality of the droplets on the coated region.

In the manufacturing method of a cell structure of Aspect (IV), in eachcoated region, the bottom area of the droplet is preferably smaller thanthe area of the coated region.

In the manufacturing method of a cell structure of Aspect (IV), thenumber of cells included in the droplet is preferably 3.0×10⁵ cells/mLor less.

In the manufacturing method of a cell structure of Aspect (IV), thedroplet preferably has a diameter of 1 μm to 8 mm.

In the manufacturing method of a cell structure of Aspect (IV), theamount of the droplet is preferably 0.5 μL to 50 μL.

The following is a summary of Aspect (V).

A manufacturing method of a cell structure of Aspect (V) includes apreparation step of preparing, on a culturing surface of a cell culturecontainer, a first coated region coated with a temperature-responsivepolymer and/or a temperature-responsive polymer composition, and aplurality of second coated regions located at an edge of the firstcoated region and coated with a cell adhesive substance, and a seedingand culturing step of seeding cells in the first coated region and thesecond coated regions and culturing the cells to produce a cellstructure.

In the manufacturing method of a cell structure of Aspect (V), theculturing surface is preferably cell non-adhesive.

In the manufacturing method of a cell structure of Aspect (V), the celladhesive substance is preferably at least one selected from the groupconsisting of laminin, collagen, and fibronectin.

In the manufacturing method of a cell structure of Aspect (V), theregion occupied by the first coated region and the second coated regionsis preferably surrounded by a cell non-adhesive wall.

In the manufacturing method of a cell structure of Aspect (V), the firstcoated region preferably has a shape extending in a predetermineddirection, and the edge of the first coated region preferably lies inthe predetermined direction.

A cell structure of Aspect (V) is manufactured using any of theaforementioned manufacturing methods of a cell structure.

A cell of Aspect (V) includes, on a culturing surface, a first coatedregion coated with a temperature-responsive polymer and/or atemperature-responsive polymer composition, and a plurality of secondcoated regions located at an edge of the first coated region and coatedwith a cell adhesive substance.

Aspect (VI) provides a manufacturing method of a cell structure, themanufacturing method including a production step of producing atemperature-responsive polymer or a temperature-responsive polymercomposition, a culture container preparation step of coating a culturingsurface of a cell culture container with the temperature-responsivepolymer or the temperature-responsive polymer composition to prepare acoated cell culture container, a seeding step of seeding cardiomyocytesand fibroblasts in the coated cell culture container at a ratio of 200to 300 fibroblasts per 100 cardiomyocytes, and a culturing step ofculturing the seeded cells to obtain an aggregated cell structure.

In the seeding step, vascular endothelial cells are preferably furtherseeded.

Immune system cells are preferably added to the coated cell culturecontainer during or after the seeding step and before obtaining the cellstructure.

The immune system cells are preferably macrophages and/or T cells.

The area of the portion coated with the temperature-responsive polymeror the temperature-responsive polymer composition is preferably 200 mm²or less.

Aspect (VII) provides a manufacturing method of a cell structure, themanufacturing method including a production step of producing atemperature-responsive polymer or a temperature-responsive polymercomposition, a culture container preparation step of coating a culturingsurface of a cell culture container with the temperature-responsivepolymer or the temperature-responsive polymer composition to prepare acoated cell culture container, a seeding step of seeding hepatocytes andfibroblasts in the coated cell culture container at a ratio of 10 to 50fibroblasts per 100 hepatocytes, and a culturing step of culturing theseeded cells to obtain an aggregated cell structure.

In the seeding step, vascular endothelial cells are preferably furtherseeded.

In the seeding step, adipocytes are preferably further seeded.

In the seeding step, the adipocytes are preferably seeded in a ratio of50 adipocytes per 100 hepatocytes and 100 to 500 adipocytes per 100fibroblasts.

Immune system cells are preferably added to the coated cell culturecontainer during or after the seeding step and before obtaining the cellstructure.

The immune system cells are preferably at least one selected from thegroup consisting of monocytes, granulocytes, lymphocytes, andmacrophages.

Advantageous Effect

The present disclosure can provide a method of efficiently manufacturinga cell mass, a cell structure, or a three-dimensional tissue body.

In particular, Aspect (I) allows easy manufacturing of a chondrocytemass and a graft material, along with a composite material, that areuseful for treatment of joints, the trachea, the nose, and the like.

The culture method of epithelial cells in Aspect (II) has theaforementioned configuration, thereby allowing epithelial cells thattend not to adhere to a cell culture container to be cultured easily.The manufacturing method of a cell structure in Aspect (II) has theaforementioned configuration, thereby allowing a cell structureincluding epithelial cells that tend not to adhere to a cell culturecontainer to be manufactured easily. The cell culture container forepithelial cells in Aspect (II) has the aforementioned structure,thereby allowing the culturing of epithelial cells and the manufacturingof a cell structure thereof.

The production apparatus of a three-dimensional tissue body in Aspect(III) has the aforementioned configuration, thereby allowing easyproduction of a three-dimensional tissue body with a ringed shape, aluminal shape, or the like. The production method of a three-dimensionaltissue body in Aspect (III) has the aforementioned configuration,thereby allowing easy production of a three-dimensional tissue body witha ringed shape, a luminal shape, or the like.

Furthermore, Aspect (IV) allows easy manufacturing of cell structureshaving a desired size and a well-defined spheroidal shape.

Aspect (V) can control the aggregation mode of cells to manufacture cellstructures with a desired form.

The manufacturing method of a cell structure in Aspect (VI) has theaforementioned configuration, thereby allowing easy formation of a cellstructure that includes cardiomyocytes and fibroblasts and is useful asa heart disease model.

The manufacturing method of a cell structure in Aspect (VII) has theaforementioned configuration, thereby allowing easy formation of a cellstructure that includes hepatocytes and fibroblasts and is useful as ahepatic failure model.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is an overview, in (i) to (viii), of an example manufacturingmethod of a chondrocyte mass in Embodiment (I);

FIG. 2 is an overview, in (i) to (vi), of an example manufacturingmethod of a chondrocyte mass in Embodiment (I), and includes across-sectional view of the structure in parentheses in (iv);

FIGS. 3A and 3B are an outline of a modification to the preparation stepin Embodiment (I), with the subsequent seeding and culturing step, whereFIG. 3A illustrates a first modification to the preparation step, andFIG. 3B illustrates a second modification to the preparation step;

FIG. 4 is an overview of a manufacturing method of a graft material inEmbodiment (I);

FIG. 5 is an overview of another example manufacturing method of achondrocyte mass in Embodiment (I);

FIG. 6 is an overview, in (i) to (v), of an example manufacturing methodof a composite material in Embodiment (I);

FIG. 7 contains photographs taken when using a fluorescence microscopeto observe the state of a cell structure 24 hours (1 day), 2 days, 6days, and 10 days after the start of culturing in Test I-C-1 (ReferenceExample I) of Embodiment (I); in particular, the lower portionillustrates a partial enlargement of the photograph of the cellstructure after 10 days;

FIG. 8 is a photograph taken after cutting a cross-section of the cellstructure obtained in Test I-C-1 (Reference Example I) of Embodiment (I)along the short-axis;

FIG. 9 contains photographs taken when using a fluorescence microscopeto observe the state of a cell structure 27 hours, 44 hours, 70 hours,and 122 hours after the start of culturing in Test I-C-2 of Embodiment(I); the upper portion illustrates the state of the cell structure whenusing a pad with a 2 mm wide donut-shaped cutout, and the lower portionillustrates the state of the cell structure when using a pad with a 2.5mm wide donut-shaped cutout;

FIG. 10 contains photographs taken when using a fluorescence microscopeto observe the state of a cell structure 8 hours, 20 hours, 32 hours,and 42 hours after the start of culturing in Test I-C-3 of Embodiment(I); the upper portion illustrates the state of the cell structure whenusing a pad with a 2 mm wide donut-shaped cutout, the lower portionillustrates the state of the cell structure when using a pad with a 2.5mm wide donut-shaped cutout, and the lowermost portion is a photographtaken when using a stereomicroscope to observe the state of the cellstructure after 42 hours;

FIG. 11A is a photograph taken when observing the state of a compositematerial with the naked eye 6 days after the start of culturing in TestI-D of Embodiment (I), and FIG. 11B is a photograph taken when observingthe state of a composite material with the naked eye 21 days after thestart of culturing in Test I-D of Embodiment (I);

FIG. 12 is an enlargement of a photograph taken when observing the stateof a portion of a composite material with the naked eye 21 days afterthe start of culturing in Test I-D of Embodiment (I);

FIGS. 13A to 13D are photographs taken when observing, with the nakedeye, the state of a composite material produced in Test I-D ofEmbodiment (I) when manipulating the composite material with tweezers,where FIG. 13A illustrates the outer peripheral surface withoutmanipulation, FIG. 13B illustrates the luminal surface withoutmanipulation, FIG. 13C illustrates the state when the entire material iscrushed, and FIG. 13D illustrates the state when pulling towards aportion of the side surface;

FIGS. 14A to 14D are photographs taken when using a microscope toobserve the state of a composite material produced in Test I-D ofEmbodiment (I) when subjecting the composite material to a hematoxylinand eosin stain (H&E stain), where FIG. 14A is an exterior photograph ofthe composite material, FIG. 14B is a cross-sectional view of thecomposite material in a plane along the A-A line in FIG. 14A, and FIGS.14C and 14D are partial enlargements of the photograph in FIG. 14B;

FIG. 15 is an outline illustrating a manufacturing method of a cellstructure in an embodiment of Aspect (II);

FIG. 16 is an outline illustrating a manufacturing method of a cellstructure in an embodiment of Aspect (II);

FIG. 17 is a photograph illustrating the state when culturing epithelialcells for 96 hours on a temperature-responsive polymer or atemperature-responsive polymer composition used in Aspect (II); theportion surrounded by the dashed line indicates a coated region A, thearrow with a black outline indicates cells adhered to and growing in thecoated region A, and the solid black arrows indicate cells adhered toand growing in the non-coated region;

FIG. 18 is an outline (perspective view) illustrating a productionapparatus of a three-dimensional tissue body in an embodiment of Aspect(III);

FIG. 19 is an outline (perspective view) illustrating a productionapparatus of a three-dimensional tissue body in an embodiment of Aspect(III);

FIG. 20 is an outline (perspective view) illustrating a productionapparatus of a three-dimensional tissue body in an embodiment of Aspect(III);

FIG. 21 is a photograph of a production apparatus of a three-dimensionaltissue body in an embodiment of Aspect (III);

FIG. 22 is an outline illustrating a production method of athree-dimensional tissue body in an embodiment of Aspect (III);

FIG. 23 is an outline illustrating a production method of athree-dimensional tissue body in an embodiment of Aspect (III);

FIG. 24 is a photograph of a ringed three-dimensional tissue bodyobtained in Example III-4 of Aspect (III);

FIG. 25 is a photograph of a luminal three-dimensional tissue bodyobtained in Example III-5 of Aspect (III);

FIG. 26 is an outline (perspective view) illustrating a productionapparatus of a three-dimensional tissue body in an embodiment of Aspect(III);

FIG. 27 is an outline illustrating a production method of athree-dimensional tissue body in an embodiment of Aspect (III);

FIG. 28A is a photograph of a luminal three-dimensional tissue bodyobtained in Example III-8 of Aspect (III), and FIG. 28B is an HE stainsection image of a luminal three-dimensional tissue body obtained inExample III-8 of Aspect (III);

FIG. 29 is a photograph of a synthetic blood vessel obtained in ExampleIII-9 of Aspect (III);

FIG. 30 is a photograph of a synthetic trachea obtained in ExampleIII-10 of Aspect (III);

FIG. 31 is a photograph of a three-dimensional tissue body, havingprotein as the principal component, obtained in Example III-11 of Aspect(III);

FIG. 32 is an outline of an example manufacturing method of a cellculture body in an embodiment of Aspect (IV);

FIG. 33 illustrates a modification, in (i) to (iii), to themanufacturing method of a cell culture body in an embodiment of Aspect(IV);

FIGS. 34A to 34C illustrate the results of investigating the correlationbetween the amount of a droplet and the diameter of the droplet on theculturing surface in an example of Aspect (IV);

FIG. 35 is an overview, in (i) to (viii), of an example manufacturingmethod of a cell structure in Embodiment (V);

FIGS. 36A to 36C illustrate arrangements of a first coated region and afirst coated region in Embodiment (V);

FIG. 37 is an outline of a modification to the preparation step inEmbodiment (V), with the subsequent seeding and culturing step;

FIG. 38A is a photograph when using a microscope, in Test V-C inEmbodiment (V), to observe the state after 2 hours of culturing ADSC ofa GFP recombinant Lewis rat in the first coated region and the secondcoated regions prepared in Test V-B; FIG. 38B is a photograph when usinga microscope, in Test V-C, to observe the state after 20 hours ofculturing ADSC of a GFP recombinant Lewis rat in the first coated regionand the second coated regions prepared in Test V-B; FIG. 38C is aphotograph when observing the cell structure in FIG. 38B at lowermagnification; and FIG. 38D is a photograph when using a fluorescencemicroscope to observe the state of the cell structure indicated by thedashed line in FIG. 38B;

FIG. 39 is an outline illustrating a manufacturing method of a cellstructure in an embodiment of Aspect (VI);

FIG. 40 is an outline illustrating a manufacturing method of a cellstructure in an embodiment of Aspect (VI);

FIG. 41 is an outline illustrating a manufacturing method of a cellstructure in an embodiment of Aspect (VII); and

FIG. 42 is an outline illustrating a manufacturing method of a cellstructure in an embodiment of Aspect (VII).

DETAILED DESCRIPTION

In the present disclosure, cells are seeded and cultured on a culturingsurface coated with a temperature-responsive polymer or atemperature-responsive polymer composition. Specifically, the presentdisclosure encompasses Aspects (I) to (VII) below.

[[Aspect (I)]]

With regard to Aspect (I), we have previously developed atemperature-responsive polymer and a temperature-responsive polymercomposition that have particular properties and are extremely useful formanufacturing cell structures. When a culturing surface of a cellculture container is coated with this polymer and/or polymer compositionand cells corresponding in number to a confluency of approximately 100%are seeded and cultured, the cells adhere to the coating andsubsequently aggregate at once on the coated culturing surface to form acell mass in the central portion of the coated culturing surface. Thisphenomenon is thought to be due to contraction by the inter-cell networkexceeding adhesion of the cells to the coated culturing surface, causingthe cells to separate from the coated culturing surface.

The manufacturing method of a chondrocyte mass and a graft material inAspect (I) uses this temperature-responsive polymer ortemperature-responsive polymer composition.

With reference to the drawings, embodiments of the manufacturing methodof a chondrocyte mass and a graft material in Aspect (I) and of thechondrocyte mass and graft material in Aspect (I) are described indetail with examples.

(Manufacturing Method of Chondrocyte Mass)

A manufacturing method of a chondrocyte mass in an embodiment(Embodiment (I)) of Aspect (I) includes a seeding and culturing step ofseeding, in the presence of a cell mass, cells capable ofdifferentiating into chondrocytes onto a coated culturing surface coatedwith a temperature-responsive polymer or a temperature-responsivepolymer composition and coculturing the cell mass and the cells capableof differentiating into chondrocytes to produce a chondrocyte mass.

The manufacturing method of Embodiment (I) preferably includes aproduction step of producing a temperature-responsive polymer and/or atemperature-responsive polymer composition, a preparation step ofcoating a portion of a culturing surface with the temperature-responsivepolymer and/or the temperature-responsive polymer composition to preparea coated culturing surface, and a seeding and culturing step of seeding,in the presence of a cell mass, cells capable of differentiating intochondrocytes onto the coated culturing surface and coculturing the cellmass and the cells capable of differentiating into chondrocytes toproduce a chondrocyte mass.

An example manufacturing method of a chondrocyte mass in Embodiment (I)is outlined in (i) to (viii) of FIG. 1 and (i) to (vi) of FIG. 2.

Details of each step in an example manufacturing method of a chondrocytemass in Embodiment (I) are provided below.

(Production Step)

In an example manufacturing method, a temperature-responsive polymerand/or a temperature-responsive polymer composition is first produced(production step).

Examples of the temperature-responsive polymer andtemperature-responsive polymer composition include (A) atemperature-responsive polymer containing 2-N,N-dimethylaminoethylmethacrylate (DMAEMA) units and anionic monomer units, (B) atemperature-responsive polymer containing N-isopropyl acrylamide (NIPAM)units, cationic monomer units, and anionic monomer units, and (C) atemperature-responsive polymer composition containing a polymer of2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and/or a derivativethereof, 2-amino-2-hydroxymethyl-1,3-propanediol (tris), and one or moreanionic substances selected from the group consisting of nucleic acids,heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid,polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfatedpolysaccharide, curdlan, polyarginic acid, and alkali metal saltsthereof. Examples of (A) include (A-1) a temperature-responsive polymerobtained by a method of polymerizing DMAEMA in the presence of water and(A-2) a temperature-responsive polymer containing a polymer blockprincipally containing DMAEMA (polymer chain α terminal) and a copolymerblock principally containing DMAEMA and an anionic monomer (polymerchain ω terminal).

One type of these polymers and polymer compositions may be used alone,or a combination of two or more types may be used in Embodiment (I).

The temperature-responsive polymer of (A-1) and a manufacturing methodthereof are described below.

(Manufacturing Method of Temperature-Responsive Polymer)

A manufacturing method of the temperature-responsive polymer of (A-1)includes a production step of producing a mixture containing2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and an irradiation stepof irradiating the mixture with ultraviolet light, where in theproduction step, the mixture further contains a polymerization inhibitorand water, and in the irradiation step, the mixture is irradiated withultraviolet light under an inert atmosphere.

In a manufacturing method of the temperature-responsive polymer of(A-1), a mixture containing 2-N,N-dimethylaminoethyl methacrylate(DMAEMA) is first produced (production step). The mixture furtherincludes a polymerization inhibitor and water.

A commercial product may be used as the 2-N,N-dimethylaminoethylmethacrylate (DMAEMA). Examples of the polymerization inhibitor includemethylhydroquinone (MEHQ), hydroquinone, p-benzoquinoline,N,N-diethylhydroxylamine, N-nitroso-N-phenylhydroxylamine (Cupferron),and t-butylhydroquinone. MEHQ or the like included in commerciallyavailable DMAEMA may be used as is. Examples of water include ultrapurewater.

The mass ratio of the polymerization inhibitor to the mixture ispreferably 0.01% to 1.5% and more preferably 0.1% to 0.5%. Adoptingthese ranges suppresses a runaway radical polymerization reaction andreduces the occurrence of uncontrollable crosslinking, while alsoproviding the manufactured temperature-responsive polymer withsolubility in a solvent.

The mass ratio of the water to the mixture is preferably 1.0% to 50% andmore preferably 9.0% to 33%. Adopting these ranges achieves a goodbalance between the reaction rate of the hydrolysis reaction of the sidechain and the reaction rate of the growth reaction of the polymer chainbeing polymerized. It is thus possible to obtain atemperature-responsive polymer having a ratio of DMAEMA in which theside chain is not hydrolyzed to DMAEMA in which the side chain ishydrolyzed (the copolymerization ratio) of approximately 1.0 to 20.

Next, in the manufacturing method of the temperature-responsive polymerof (A-1), the mixture is irradiated with ultraviolet light (irradiationstep). Here, the irradiation with ultraviolet light takes place under aninert atmosphere. The DMAEMA undergoes radical polymerization byirradiation with ultraviolet light to become a polymer.

In this step, the aforementioned mixture is added to a transparent,sealed vial, for example, and an inert atmosphere is formed inside thevial by bubbling an inert gas. Subsequently, the mixture is irradiatedwith ultraviolet light from outside the vial using an ultraviolet lightirradiation apparatus.

The wavelength of the ultraviolet light is preferably 210 nm to 600 nmand more preferably 360 nm to 380 nm. These wavelength ranges can causethe polymerization reaction to progress efficiently and stably yieldpolymer material with the desired copolymerization ratio. Thesewavelength ranges can also prevent coloring of the manufactured polymermaterial.

Examples of the inert gas include nitrogen, argon, helium, and neon.

Among reaction conditions, the temperature condition is preferably from15° C. to 50° C., more preferably from 20° C. to 30° C. Thesetemperature ranges suppress a heat initiated reaction, giving preferenceinstead to a reaction initiated by irradiation with light. Furthermore,the reaction rate of the hydrolysis reaction can be balanced wellagainst the reaction rate of the growth reaction of the polymer chain.

The reaction time is preferably from 7 hours to 24 hours, morepreferably from 17 hours to 21 hours. These time ranges can obtain ahigh yield of the temperature sensitive polymer of (A-1) and allowradical polymerization while suppressing a photolytic reaction and anunnecessary crosslinking reaction.

The time from when production of the mixture in the production step isfinished until the start of irradiation with ultraviolet light in theirradiation step is preferably from 10 minutes to 1 hour.

It takes approximately 10 minutes to replace the gas inside the vial towhich the mixture is added and to form an inert atmosphere inside thevial. Setting the aforementioned time to less than 10 minutes maytherefore not result in the inert atmosphere necessary for radicalpolymerization. On the other hand, the hydrolysis reaction of DMAEMA inthe mixture starts before the start of irradiation with ultravioletlight. Setting the aforementioned time to longer than one hour thereforeyields a large amount of methacrylic acid, which is inactive in theradical polymerization reaction, in the mixture.

In the manufacturing method of the temperature-responsive polymer of(A-1), water is included in the mixture. The radical polymerizationreaction of DMAEMA and the hydrolysis reaction of the ester bond in theside chain of the poly(2-N,N-dimethylaminoethyl methacrylate) (PDMAEMA)can therefore be caused to compete.

The product yielded by this competition is a polymer including therepeating unit (A) represented by formula (I),

and the repeating unit (B) represented by formula (II).

Therefore, a good balance of both the cationic functional group includedin the polymer, i.e. a dimethylamino group, and the anionic functionalgroup included in the polymer, i.e. a carboxyl group formed byhydrolysis of the ester bond in a side chain, can be provided. Themanufacturing method of the temperature-responsive polymer of (A-1) canthen easily manufacture, with few steps, a polymer derived frompoly(2-N,N-dimethylaminoethyl methacrylate) and including a cationicfunctional group and an anionic functional group.

Even without using the same manufacturing method as the manufacturingmethod of the temperature-responsive polymer of (A-1), the same effectsas those of the manufacturing method of a temperature-responsive polymerof Aspect (I) may be obtained if DMAEMA, a polymerization inhibitor, andwater are present together in the reaction system at the time ofirradiating with ultraviolet light.

For example, the following manufacturing method of atemperature-responsive polymer can also be used for thetemperature-responsive polymer of (A-1): water and a mixture containingDMAEMA and a polymerization inhibitor are prepared separately, an inertgas is then bubbled in the mixture and the water, and subsequently, themixture and the water are mixed under an inert atmosphere whilesimultaneously being irradiated with ultraviolet light.

(Temperature-Responsive Polymer)

The temperature-responsive polymer of (A-1) is manufactured by theaforementioned manufacturing method of (A-1).

The temperature-responsive polymer of (A-1) is preferably a moleculewith a number-average molecular weight (Mn) of 10 kDa to 500 kDa. Thetemperature-responsive polymer of (A-1) is also preferably a moleculefor which the ratio (Mw/Mn) of the weight-average molecular weight (Mw)to the number-average molecular weight (Mn) is from 1.1 to 10.0.

The molecular weight of the temperature-responsive polymer of (A-1) canbe appropriately adjusted by the irradiation time and irradiationintensity of the ultraviolet light.

The temperature-responsive polymer of (A-1) can reduce the cloud point,for example to room temperature (25° C.) or below.

Insoluble matter of the temperature-responsive polymer (A-1) formed at atemperature at or above the cloud point exhibits an extremely long delayuntil becoming soluble again at room temperature (approximately 25° C.).The reason is thought to be that the resulting temperature-responsivepolymer of (A-1) has high self-cohesion due to the presence of acationic functional group and an anionic functional group in themolecule.

As described below, the temperature-responsive polymer of (A-1) can beused to produce a cell culture container having a culturing surfacecoated with this temperature-responsive polymer.

Furthermore, as described below, the temperature-responsive polymer of(A-1) allows formation of cell structures that have a luminal(tube-like), aggregated (pellet-like), or other structure by culturingcells under appropriate culture conditions.

The ratio (C/A ratio) of the number of cationic functional groups(2-N,N-dimethylamino groups) to the number of anionic functional groups(carboxyl groups) in the temperature-responsive polymer of (A-1) ispreferably from 0.5 to 32 and more preferably from 4 to 16.

Setting the C/A ratio in these ranges facilitates achievement of theaforementioned effect of reducing the cloud point. The reason is thoughtto be that in a temperature-responsive polymer with the aforementionedC/A ratio, the cationic functional group and the anionic functionalgroup affect inter- and/or intra-molecular aggregation by ionic bondingthroughout the temperature-responsive polymer, thereby increasing theaggregation strength of the temperature-responsive polymer.

Another reason is thought to be that setting the C/A ratio within theaforementioned ranges can suppress cytotoxicity due to positive chargesby achieving a particularly preferable balance between positive andnegative charges in the temperature-responsive polymer and can alsofacilitate cell migration and orientation by achieving a particularlypreferable balance between hydrophilicity and hydrophobicity of thetemperature-responsive polymer.

The temperature-responsive polymer of (A-2) and a manufacturing methodthereof are described below.

(Manufacturing Method of Temperature-Responsive Polymer)

A manufacturing method of the temperature-responsive polymer of (A-2)includes a first polymerization step of irradiating a first mixturecontaining 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) withultraviolet light, an adding step of adding an anionic monomer to thefirst mixture at the point when the number-average molecular weight ofthe polymer in the first polymerization step reaches at least apredetermined value to produce a second mixture, and a secondpolymerization step of irradiating the second mixture with ultravioletlight.

In a manufacturing method of the temperature-responsive polymer of(A-2), the first mixture containing 2-N,N-dimethylaminoethylmethacrylate (DMAEMA) is first irradiated with ultraviolet light (firstpolymerization step).

Other than DMAEMA, the first mixture may, for example, optionallyinclude another monomer, solvent, or the like.

The irradiation with ultraviolet light may take place under an inertatmosphere.

A commercially available product may be used for the DMAEMA.

Examples of the other monomers that may be included in the first mixtureinclude N,N-dimethyl acrylamide, esters of acrylic acid or methacrylicacid having polyethylene glycol side chains, N-isopropyl acrylamide,3-N,N-dimethylaminopropyl acrylamide, and 2-N,N-dimethylaminoethylmethacrylamide. In particular, N,N-dimethyl acrylamide, esters ofacrylic acid or methacrylic acid having polyethylene glycol side chains,and N-isopropyl acrylamide are preferable for allowing the ion balanceto be adjusted stably. One type of these monomers may be used alone, ora combination of two or more types may be used. The ratio (mole ratio)of the amount of other monomers used to the amount of DMAEMA used ispreferably from 0.001 to 1 and more preferably from 0.01 to 0.5.

Examples of the solvent include toluene, benzene, chloroform, methanol,and ethanol. In particular, toluene and benzene are preferable by virtueof being inert relative to the ester bond of the DMAEMA. One type ofthese solvents may be used alone, or a combination of two or more typesmay be used.

In this step, the aforementioned first mixture is added to atransparent, sealed vial, for example, and an inert atmosphere is formedinside the vial by bubbling an inert gas. Subsequently, the firstmixture is irradiated with ultraviolet light from outside the vial usingan ultraviolet light irradiation apparatus.

The wavelength of the ultraviolet light is preferably 210 nm to 600 nmand more preferably 360 nm to 380 nm. These wavelength ranges can causethe polymerization reaction to progress efficiently and stably yieldpolymer material with the desired copolymerization ratio. Thesewavelength ranges can also prevent coloring of the manufactured polymermaterial.

The irradiation intensity of the ultraviolet light is preferably from0.01 mW/cm² to 50 mW/cm² and more preferably from 0.1 mW/cm² to 5mW/cm². These ranges can suppress decomposition due to unnecessarycutting of chemical bonds or the like while stably allowing thepolymerization reaction to proceed at an appropriate rate (time).

Examples of the inert gas include nitrogen, argon, helium, and neon.

The temperature condition is preferably from 10° C. to 40° C., morepreferably from 20° C. to 30° C. These temperature ranges allow thereaction to take place at room temperature in a typical laboratory whilesuppressing a reaction due to means other than light (such as heat).

The reaction time is preferably from 10 minutes to 48 hours, morepreferably from 60 minutes to 24 hours.

In this step, the DMAEMA undergoes radical polymerization by irradiationwith the ultraviolet light and becomes a polymer(poly(2-N,N-dimethylaminoethyl methacrylate), i.e. PDMAEMA), therebyforming a homopolymer block containing 2-N,N-dimethylaminoethylmethacrylate. In the case of also using another monomer, a polymer blockcontaining DMAEMA and the other monomer is formed.

Next, in the manufacturing method of the temperature-responsive polymerof (A-2), at the point when the number-average molecular weight of thepolymer (specifically, polymerized 2-N,N-dimethylaminoethylmethacrylate) reaches at least a predetermined value in the firstpolymerization step, an anionic monomer is added to the first mixture toproduce a second mixture (adding step).

Other than the first mixture after the first polymerization step and theanionic monomer, the second mixture may, for example, include anothermonomer, the above-described solvents that can be included in the firstmixture (such as toluene, benzene, or methanol), and the like.

The anionic monomer may be added under an inert atmosphere.

Examples of the anionic monomer include acrylic acid, methacrylic acid,and vinyl derivatives containing at least one group selected from thegroup consisting of a carboxyl group, a sulfonic acid group, and aphosphoric acid group in a side chain. In particular, acrylic acid andmethacrylic acid are preferable in terms of chemical stability.

One type of these anionic monomers may be used alone, or a combinationof two or more types may be used.

Examples of the other monomers that may be included in the secondmixture include N,N-dimethyl acrylamide, esters of acrylic acid ormethacrylic acid having polyethylene glycol side chains, N-isopropylacrylamide, 3-N,N-dimethylaminopropyl acrylamide, and2-N,N-dimethylaminoethyl methacrylamide. N,N-dimethyl acrylamide, whichis electrically neutral and hydrophilic, is particularly preferable. Onetype of these monomers may be used alone, or a combination of two ormore types may be used. The ratio (in moles) of the amount of othermonomers used to the amount of DMAEMA used is preferably from 0.01 to 10and more preferably from 0.1 to 5.

In this step, the second mixture is added while, for example,maintaining an inert atmosphere in the vial by causing an inert gas toflow into the vial.

The predetermined value of the number-average molecular weight ispreferably 5,000, more preferably 20,000, and particularly preferably100,000 to sufficiently obtain the effect of reducing the cloud point.

The number-average molecular weight of the polymerized PDMAEMA in thefirst mixture after the first polymerization step can be measured bysampling a small amount of the reaction mixture from the polymerizationsystem at a predetermined point in time and using a method known to aperson skilled in the art, such as gel permeation chromatography (GPC)or static light scattering (SLS).

In this step, an anionic monomer is included in the polymerizationsystem in addition to the homopolymer containing DMAEMA that is beingpolymerized. The polymerization system in the vial thereby changes froma homopolymerization system of DMAEMA to a copolymerization system ofDMAEMA and an anionic monomer.

In the manufacturing method of the temperature-responsive polymer of(A-2), the second mixture is then irradiated with ultraviolet light(second polymerization step).

Here, the irradiation with ultraviolet light may take place under aninert atmosphere.

During this step, the vial to which the second mixture has been addedis, for example, irradiated with ultraviolet light from outside the vialusing an ultraviolet light irradiation apparatus.

The conditions in the second polymerization step, such as the wavelengthof the ultraviolet light, the radiation intensity of the ultravioletlight, the inert gas that is used, the reaction temperature, and thereaction time may be the same as the conditions in the firstpolymerization step.

In this step, the DMAEMA and the anionic monomer undergo radicalpolymerization by irradiation with the ultraviolet light, and acopolymer block containing DMAEMA and the anionic monomer is formed tobe continuous with the polymer chain α terminal of the homopolymerblock, which contains DMAEMA, formed in the first polymerization step.In the case of also using another monomer, a copolymer block containingDMAEMA, an anionic monomer, and the other monomer is formed.

As described above, a temperature-responsive polymer containing ahomopolymer block containing DMAEMA and a copolymer block of DMAEMA andan anionic monomer is obtained.

As will be understood by a person skilled in the art, while mixtures ofpolymers having various molecular weights and molecular structures aregenerated with the manufacturing method of (A-2), polymerization ispreferably carried out under identical conditions throughout the firstpolymerization step, the adding step, and the second polymerization stepto obtain, as the principal component, a temperature-responsive polymercontaining a homopolymer block containing DMAEMA and a copolymer blockof DMAEMA and an anionic monomer.

(Temperature-Responsive Polymer)

The temperature-responsive polymer of (A-2) is manufactured by theaforementioned manufacturing method of (A-2).

The temperature-responsive polymer of (A-2) contains a polymer block(polymer chain α terminal) principally containing2-N,N-dimethylaminoethyl methacrylate and optionally containing othermonomer units such as dimethyl acrylamide, acrylic acid or methacrylicacid having polyethylene glycol side chains, or another such hydrophilicmonomer; and contains a copolymer block principally containing2-N,N-dimethylaminoethyl methacrylate and an anionic monomer (polymerchain ω terminal) and optionally containing other monomer units.

The temperature-responsive polymer of (A-2) preferably contains ahomopolymer block of DMAEMA and a copolymer block of DMAEMA and ananionic monomer, and the temperature-responsive polymer of (A-2) is morepreferably composed of these blocks.

As the temperature-responsive polymer of (A-2), the number-averagemolecular weight of the polymer block of the polymer chain α terminal(for example, the homopolymer block of DMAEMA) is preferably 5,000 Da orgreater and more preferably 20,000 Da or greater.

The temperature-responsive polymer of (A-2) is preferably a moleculewith a number-average molecular weight (Mn) of 10 kDa to 500 kDa. Thetemperature-responsive polymer of (A-2) is also preferably a moleculefor which the ratio (Mw/Mn) of the weight-average molecular weight (Mw)to the number-average molecular weight (Mn) is 1.1 to 10.0.

The molecular weight of the temperature-responsive polymer can beappropriately adjusted by the irradiation time and irradiation intensityof the ultraviolet light.

The temperature-responsive polymer of (A-2) can reduce the cloud point,for example to room temperature (25° C.) or below.

Insoluble matter of the temperature-responsive polymer (A-2) formed at atemperature at or above the cloud point exhibits an extremely long delayuntil becoming soluble again at room temperature (approximately 25° C.).The reason is thought to be that the resulting temperature-responsivepolymer has high self-cohesion due to the presence of a cationicfunctional group and an anionic functional group in the molecule.

In particular, it is thought that since the temperature-responsivepolymer of (A-2) includes a homopolymer block of DMAEMA having a highmolecular weight (such as 5,000 Da or greater) at the polymer chain αterminal, temperature dependent globule transition of the side chain ofDMAEMA occurs more easily, effectively reducing the cloud point.

As described below, this temperature-responsive polymer can be used toproduce a cell culture container having a culturing surface coated withthis temperature-responsive polymer.

Furthermore, as described below, the temperature-responsive polymer of(A-2) allows formation of cell structures that have a luminal(tube-like), aggregated (pellet-like), or other structure by culturingcells under appropriate culture conditions.

The ratio (C/A ratio) of the number of cationic functional groups(2-N,N-dimethylamino groups) to the number of anionic functional groups(carboxyl groups) in the temperature-responsive polymer of (A-2) ispreferably from 0.5 to 32 and more preferably from 4 to 16.

Setting the C/A ratio in these ranges facilitates achievement of theaforementioned effect of reducing the cloud point. The reason is thoughtto be that in a temperature-responsive polymer with the aforementionedC/A ratio, the cationic functional group and the anionic functionalgroup affect inter- and/or intra-molecular aggregation by ionic bondingthroughout the temperature-responsive polymer, thereby increasing theaggregation strength of the temperature-responsive polymer.

Another reason is thought to be that setting the C/A ratio within theaforementioned ranges can suppress cytotoxicity due to positive chargesby achieving a particularly preferable balance between positive andnegative charges in the temperature-responsive polymer and can alsofacilitate cell migration and orientation by achieving a particularlypreferable balance between hydrophilicity and hydrophobicity of thetemperature-responsive polymer.

The temperature-responsive polymer of (B) and a manufacturing methodthereof are described below.

(Manufacturing Method of Temperature-Responsive Polymer)

A manufacturing method of the temperature-responsive polymer of (B)polymerizes N-isopropyl acrylamide (NIPAM) (monomer (A)), a cationicmonomer (monomer (B)), and an anionic monomer (monomer (C)). Anothermonomer, other than the aforementioned 3 types of monomers, mayoptionally be added and polymerized.

A commercially available product may be used for the N-isopropylacrylamide (NIPAM).

Examples of cationic monomers include monomers having a cationicfunctional group. Examples of cationic functional groups include aminogroups, such as primary to quaternary amino groups, and guanidinegroups. In particular, tertiary amino groups are preferable for chemicalstability, low cytotoxicity, sterilization stability, and a strongpositive charge.

More specifically, the cationic monomer is preferably highly stable evenwhen supporting a physiologically active substance or under alkalineconditions. Examples include3-(N,N-dimethylaminopropyl)-(meth)acrylamide,3-(N,N-dimethylaminopropyl)-(meth)acrylate, aminostyrene,2-(N,N-dimethylaminoethyl)-(meth)acrylamide, and2-(N,N-dimethylaminoethyl)-(meth)acrylate.

Among these, 3-(N,N-dimethylaminopropyl)acrylamide is particularlypreferable for easily supporting an anionic substance by virtue ofhaving a strong positive charge.

Aminostyrene is also preferable for easily supporting an anionicsubstance by virtue of having a strong positive charge while alsoincreasing the number of variations of supportable anionic substancesthrough interaction between an aromatic ring in the molecule and ahydrophobic structure of another substance in the aqueous solution.

Furthermore, 2-(N,N-dimethylaminoethyl)-methacrylamide is preferable forhaving a weak positive charge at a neutral pH and for its solubility inwater not being affected by temperature, thereby allowing easy ejectionof an anionic substance that has been supported once.

One type of these cationic monomers may be used alone, or a combinationof two or more types may be used.

Examples of anionic monomers include monomers having an anionicfunctional group. Examples of anionic functional groups include acarboxylic acid group, a sulfonic acid group, a sulfuric acid group, aphosphoric acid group, and a boronic acid group. In particular, acarboxylic acid group, a sulfonic acid group, and a phosphoric acidgroup are preferable for chemical stability, cell affinity, and a highdegree of purification.

More specifically, examples include acrylic acid, methacrylic acid, andvinylbenzoic acid. In particular, methacrylic acid and vinylbenzoic acidare preferable for chemical stability and cell affinity.

One type of these anionic monomers may be used alone, or a combinationof two or more types may be used.

Examples of other monomers include dimethyl acrylamide, acrylic acid ormethacrylic acid having a polyethylene glycol side chain, or anothersuch neutral hydrophilic monomer.

One type of these monomers may be used alone, or a combination of two ormore types may be used.

The other monomers can be used to adjust the hydrophilic/hydrophobicbalance apart from charge and can increase the number of variations.

Taking into consideration the reactivity in the polymerization reactionof the monomers, a person skilled in the art can appropriately adjustthe ratio (moles) of the amount of NIPAM used, the amount of cationicmonomers used, and the amount of other monomers used relative to thetotal amount of monomers (A) to (C) used in the manufacturing method ofthe temperature-responsive polymer of (B) so that the desired ratio ofmonomer components is obtained.

Examples of polymerization methods include radical polymerization andionic polymerization.

Living radical polymerization is preferable as a type of radicalpolymerization. Examples of living radical polymerization includereversible addition fragmentation chain transfer (RAFT) polymerization,atom transfer radical polymerization (ATRP), and iniferterpolymerization, with iniferter polymerization being preferable.

Living anionic polymerization is preferable as ionic polymerization.

An example of the manufacturing method of the temperature-responsivepolymer of (B) is a method using radical polymerization.

An example of this manufacturing method includes a first polymerizationstep of irradiating a first mixture containing N-isopropyl acrylamide(NIPAM) with ultraviolet light, an adding step of adding a cationicmonomer and an anionic monomer to the first mixture to produce a secondmixture, and a second polymerization step of irradiating the secondmixture with ultraviolet light.

In an example of this manufacturing method, a first mixture containingN-isopropyl acrylamide (NIPAM) is first irradiated with ultravioletlight (first polymerization step).

Other than NIPAM, the first mixture may, for example, optionally includeanother monomer, a solvent, a chain transfer agent, a stabilizer, asurfactant, or the like.

The irradiation with ultraviolet light may take place under an inertatmosphere.

In this step, the aforementioned first mixture is added to atransparent, sealed vial, for example, and an inert atmosphere is formedinside the vial by bubbling an inert gas. Subsequently, the firstmixture is irradiated with ultraviolet light from outside the vial usingan ultraviolet light irradiation apparatus.

Examples of the solvent include benzene, toluene, chloroform, methanol,and water. In particular, benzene and toluene are preferable in terms ofsolubility and for being inert during polymerization. One type of thesesolvents may be used alone, or a combination of two or more types may beused.

In this step, the aforementioned first mixture is added to atransparent, sealed vial, for example, and an inert atmosphere is formedinside the vial by bubbling an inert gas. Subsequently, the firstmixture is irradiated with ultraviolet light from outside the vial usingan ultraviolet light irradiation apparatus.

The wavelength of the ultraviolet light is preferably 210 nm to 600 nmand more preferably 360 nm to 380 nm. These wavelength ranges can causethe polymerization reaction to progress efficiently and stably yieldpolymer material with the desired copolymerization ratio. Thesewavelength ranges can also prevent coloring of the manufactured polymermaterial.

The irradiation intensity of the ultraviolet light is preferably from0.01 mW/cm² to 50 mW/cm² and more preferably from 0.1 mW/cm² to 5mW/cm².

Examples of the inert gas include nitrogen, argon, helium, and neon.

The temperature condition is preferably from 10° C. to 40° C., morepreferably from 20° C. to 30° C. These temperature ranges allow thepolymerization reaction to take place at room temperature in a typicallaboratory while suppressing a reaction due to means, such as heat,differing from the light irradiation means.

The reaction time is preferably from 10 minutes to 48 hours, morepreferably from 60 minutes to 24 hours.

During this step, the NIPAM undergoes radical polymerization byirradiation with the ultraviolet light and becomes a polymer(poly(N-isopropyl acrylamide), i.e. PNIPAM), thereby forming ahomopolymer block containing N-isopropyl acrylamide. In the case of alsousing another monomer, a polymer block containing NIPAM and the othermonomer is formed.

Next, in the manufacturing method of the temperature-responsive polymerof (B), a second mixture is produced by adding a cationic monomer and ananionic monomer to the first mixture after the first polymerization step(adding step).

Other than the first mixture after the first polymerization step, thecationic monomer, and the anionic monomer, the second mixture may, forexample, include another monomer, a solvent, a chain transfer agent, astabilizer, a surfactant, or the like.

The cationic monomer and the anionic monomer may be added under an inertatmosphere.

In this step, the cationic monomer and the anionic monomer are addedwhile, for example, maintaining an inert atmosphere in the vial bycausing an inert gas to flow into the vial.

In this step, a cationic monomer and an anionic monomer are included inthe polymerization system in addition to the homopolymer containingNIPAM that is being polymerized. The polymerization system in the vialthereby changes from a homopolymerization system of NIPAM to acopolymerization system of NIPAM, a cationic monomer, and an anionicmonomer.

In the manufacturing method of the temperature-responsive polymer of(B), the second mixture is then irradiated with ultraviolet light(second polymerization step).

Here, the irradiation with ultraviolet light may take place under aninert atmosphere.

During this step, the vial to which the cationic monomer and the anionicmonomer have been added is, for example, irradiated with ultravioletlight from outside using an ultraviolet light irradiation device.

The wavelength of the ultraviolet light is preferably 210 nm to 600 nmand more preferably 360 nm to 380 nm. These wavelength ranges can causethe polymerization reaction to progress efficiently and stably yieldpolymer material with the desired copolymerization ratio. Thesewavelength ranges can also prevent coloring of the manufactured polymermaterial.

The irradiation intensity of the ultraviolet light is preferably from0.01 mW/cm² to 50 mW/cm² and more preferably from 0.1 mW/cm² to 5mW/cm².

Examples of the inert gas include nitrogen, argon, helium, and neon.

The temperature condition is preferably from 10° C. to 40° C., morepreferably from 20° C. to 30° C. These temperature ranges allow thepolymerization reaction to take place at room temperature in a typicallaboratory while suppressing a reaction due to means, such as heat,differing from the light irradiation means.

The reaction time is preferably from 10 minutes to 48 hours, morepreferably from 60 minutes to 24 hours.

In this step, the NIPAM, the cationic monomer, and the anionic monomerundergo radical polymerization by irradiation with the ultravioletlight, and a copolymer block containing NIPAM, the cationic monomer, andthe anionic monomer is formed to be continuous with the polymer chain αterminal of the homopolymer block, which includes NIPAM, formed in thefirst polymerization step. In the case of also using another monomer, apolymer block containing NIPAM and the other monomer, and/or a copolymerblock containing NIPAM, the cationic monomer, the anionic monomer, andthe other monomer is formed.

As described above, a temperature-responsive polymer containing ahomopolymer block containing NIPAM and a copolymer block of NIPAM, acationic monomer, and an anionic monomer is obtained.

In an example of this manufacturing method, irradiation with ultravioletlight is preferably performed throughout the first polymerization step,the adding step, and the second polymerization step to achieve anefficient reaction.

Another example manufacturing method of the temperature-responsivepolymer of (B) is a method using radical polymerization. A mixturecontaining N-isopropyl acrylamide (NIPAM), a cationic monomer, and ananionic monomer, and optionally containing another monomer, isirradiated with ultraviolet light.

This mixture may, for example, include a solvent, a chain transferagent, a stabilizer, a surfactant, or the like.

The irradiation with ultraviolet light may take place under an inertatmosphere.

Other conditions may be the same as in the above-described examplemanufacturing method.

Furthermore, in the case of using iniferter polymerization,benzyl-(N,N-diethyl)dithiocarbamate may be used as an iniferter andtoluene or the like used as a solvent. Living polymerization may then becarried out by irradiation with near ultraviolet light. Here, afterpolymerization by a first monomer, polymerization by a second monomercan be performed after an isolation operation, thereby yielding a blockcopolymer.

Furthermore, in the case of using ionic polymerization, an NaOH powdermay be used as the catalyst, and a solvent for reprecipitation used forpurification may be used along with an aprotic solvent as the solvent.After polymerization by a first monomer, polymerization by a secondmonomer can be performed after a reprecipitation operation (with anionic species remaining on the (o terminal after this operation),thereby yielding a block copolymer.

(Temperature-Responsive Polymer)

The temperature-responsive polymer of (B) is manufactured by theaforementioned manufacturing method of (B).

The temperature-responsive polymer of (B) contains N-isopropylacrylamide (NIPAM) units, cationic monomer units, and anionic monomerunits, and optionally contains other monomer units. This polymer can bemanufactured by the above-described example and other example of amanufacturing method.

The temperature-responsive polymer of (B) preferably contains a polymerblock (polymer chain α terminal) principally containing N-isopropylacrylamide (NIPAM) units and optionally containing other monomer unitsand a copolymer block principally containing cationic monomer units andanionic monomer units and optionally containing other monomer units. Thetemperature-responsive polymer of (B) more preferably contains ahomopolymer block of NIPAM and a copolymer block of NIPAM, a cationicmonomer, and an anionic monomer. The temperature-responsive polymer of(B) is particularly preferably composed of these blocks. This polymercan be manufactured by the above-described example manufacturing method.

In one known temperature-responsive polymer (see JP2014162865A), theDMAEMA that provides the polymer with temperature responsiveness is, atthe same time, a cationic monomer that is necessary for forming a cellstructure (along with an anionic monomer), and the DMAEMA involved intemperature responsiveness is included in the polymer chain α terminalas a polymer block.

Since a cationic monomer always exists in the polymer chain α terminalin this temperature-responsive polymer, the degree of freedom foradjusting the position of the cationic sites in the polymer chain is nothigh, and the cationic monomer is mainly limited to DMAEMA. For thesereasons, it is not necessarily easy to adjust the positive chargestrength of the cationic site or the pH of the temperature-responsivepolymer aqueous solution.

For example, in the case of using the aforementionedtemperature-responsive polymer in a drug delivery system (DDS), the typeand amount of the supportable drug may be restricted. Examples of DDStechniques include a technique of sustained release of a drug from acoated material to cells or tissue by applying a temperature-responsivepolymer supporting a drug on a cell culture container, and thenculturing cells or tissue in the cell culture container after theapplication. Since the aforementioned known temperature-responsivepolymer includes DMAEMA with a small positive charge strength, a drugthat is an anionic substance cannot always be supported easily. Hence,the type and amount of supportable drugs may be restricted.

By contrast, in the temperature-responsive polymer of (B), the NIPAMthat provides the polymer with temperature responsiveness is a neutralmonomer, and the cationic monomer that is necessary for forming a cellstructure (along with an anionic monomer) is a different monomer thanNIPAM.

In the temperature-responsive polymer of (B), a cationic monomer doesnot necessarily need to be present at the polymer chain α terminal, andthe position of the cationic site in the polymer chain can be adjustedfreely. A wide range of cationic monomers can also be used, allowing thepositive charge strength of the cationic site and the pH of thetemperature-responsive polymer aqueous solution to be adjusted easily.

When used in a drug delivery system (DDS), for example, thetemperature-responsive polymer of (B) can expand the variety ofsupportable drugs while also increasing the amount thereof. The range ofapplications of the temperature-responsive polymer can thus beincreased.

In the temperature-responsive polymer of (B), the ratio (moles) of NIPAMunits to the total of NIPAM units, cationic monomer units, and anionicmonomer units is preferably from 0.6 to 0.9, more preferably from 0.7 to0.9, and particularly preferably 0.9.

When also using another monomer, the ratio (moles) of the other monomerunits to the total of NIPAM units, cationic monomer units, and anionicmonomer units is preferably from 0.001 to 0.2 and is more preferablyfrom 0.01 to 0.1.

As the temperature-responsive polymer of (B), the number-averagemolecular weight of the polymer block of the polymer chain α terminal(for example, the homopolymer block of NIPAM) is preferably 5,000 Da orgreater and more preferably 20,000 Da or greater.

The temperature-responsive polymer of (B) is preferably a molecule witha number-average molecular weight (Mn) of 10 kDa to 500 kDa. Thetemperature-responsive polymer of (B) is also preferably a molecule forwhich the ratio (Mw/Mn) of the weight-average molecular weight (Mw) tothe number-average molecular weight (Mn) is 1.1 to 10.0.

The molecular weight of the temperature-responsive polymer can beappropriately adjusted by the polymerization conditions.

The temperature-responsive polymer of (B) can reduce the cloud point,for example to room temperature (25° C.) or below.

Insoluble matter of the temperature-responsive polymer formed at atemperature at or above the cloud point exhibits an extremely long delayuntil becoming soluble again at room temperature (approximately 25° C.).The reason is thought to be that the resulting temperature-responsivepolymer has high self-cohesion due to the presence of a cationicfunctional group and an anionic functional group in the molecule.

In particular, it is thought that since the temperature-responsivepolymer of (B) includes a homopolymer block of NIPAM having a highmolecular weight at the polymer chain α terminal, temperature dependentglobule transition of the side chain of NIPAM occurs more easily,effectively reducing the cloud point.

As described below, this temperature-responsive polymer can be used toproduce a cell culture container having a culturing surface coated withthis temperature-responsive polymer.

Furthermore, as described below, the temperature-responsive polymer of(B) allows formation of cell structures that have a luminal (tube-like),aggregated (pellet-like), or other structure by culturing cells underappropriate culture conditions.

The ratio (C/A ratio) of the number of cationic functional groups to thenumber of anionic functional groups in the temperature-responsivepolymer of (B) is preferably from 0.5 to 32 and more preferably from 4to 16.

Setting the C/A ratio in these ranges facilitates achievement of theaforementioned effect of reducing the cloud point. The reason is thoughtto be that in a temperature-responsive polymer with the aforementionedC/A ratio, the cationic functional group and the anionic functionalgroup affect inter- and/or intra-molecular aggregation by ionic bondingthroughout the temperature-responsive polymer, thereby increasing theaggregation strength of the temperature-responsive polymer.

Another reason is thought to be that setting the C/A ratio within theaforementioned ranges can suppress cytotoxicity due to positive chargesby achieving a particularly preferable balance between positive andnegative charges in the temperature-responsive polymer and can alsofacilitate cell migration and orientation by achieving a particularlypreferable balance between hydrophilicity and hydrophobicity of thetemperature-responsive polymer.

The temperature-responsive polymer of (C) and a manufacturing methodthereof are described below.

(Manufacturing Method of Temperature-Responsive Polymer Composition)

In a manufacturing method of a temperature-responsive polymercomposition of (C), a mixed-type temperature-responsive polymercomposition is first produced (mixture production step). Specifically,(C1) a polymer of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and/ora derivative thereof, (C2) 2-amino-2-hydroxymethyl-1,3-propanediol(tris), and (C3) one or more anionic substances selected from the groupconsisting of nucleic acids, heparin, hyaluronic acid, dextran sulfate,polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid,polyphosphoric acid, sulfated polysaccharide, curdlan, polyarginic acid,and alkali metal salts thereof are mixed. Note that (C2) tris is anoptional component.

(Temperature-Responsive Polymer Composition)

As described above, the temperature-responsive polymer composition of(C) includes a polymer of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA)and/or a derivative thereof; 2-amino-2-hydroxymethyl-1,3-propanediol;and one or more anionic substances selected from the group consisting ofnucleic acids, heparin, hyaluronic acid, dextran sulfate, polystyrenesulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoricacid, sulfated polysaccharide, curdlan, polyarginic acid, and alkalimetal salts thereof.

The (C1) polymer of DMAEMA and/or a derivative thereof is atemperature-responsive polymer with a cloud point of 32° C. It isinferred that the (C2) tris has the function of slightly reducing thecloud point and/or reducing the speed at which a polymer formed at ahigher temperature than the cloud point becomes soluble again whencooled to the cloud point or lower. It is also inferred that the (C2)tris has the function of stimulating cells by a positive charge derivedfrom an amino group while maintaining hydrophilicity even in ahydrophobized polymer layer. It is inferred that the (C3) anionicsubstance has the function of allowing migration and orientation of thecultured cells and of suppressing cytotoxicity.

This mixed-type temperature-responsive polymer composition can reducethe cloud point to room temperature (25° C.) or below.

In the aforementioned composition, it is inferred that the side chain ofthe polymer of DMAEMA and/or a derivative thereof and the tris interactwith each other (for example, by crosslinking), making it easier for thepolymer to aggregate.

In (C1), the polymer of DMAEMA and/or a derivative thereof is preferablya molecule with a number-average molecular weight (Mn) of 10 kDa to 500kDa. The temperature-responsive polymer of (C1) is also preferably amolecule for which the ratio (Mw/Mn) of the weight-average molecularweight (Mw) to the number-average molecular weight (Mn) is 1.1 to 6.0.

Examples of the (C1) derivative of DMAEMA include a derivative in whicha hydrogen atom of the methyl group of methacrylate is replaced by ahalogen atom, a derivative in which the methyl group of methacrylate isreplaced by a lower alkyl group, a derivative in which the hydrogen atomof the methyl group of a dimethylamino group is replaced by a halogenatom, and a derivative in which the methyl group of a dimethylaminogroup is replaced by a lower alkyl group.

The (C2) tris is preferably is a pure substance with a 99.9% or higherpurity or is a tris aqueous solution that is made neutral or basic atthe time of use, for example by addition of an alkaline substance. Whenusing tris in its commercially available state of hydrochloride, the pHof the tris aqueous solution lowers, and the cloud point of thecomposition ends up rising to approximately 70° C. Therefore, a trisaqueous solution is not preferred.

Among the examples of anionic substances listed above in (C3), examplesof the nucleic acids include DNA, RNA, and synthetic nucleic acids suchas single-stranded, double-stranded, oligomer, and hairpin nucleicacids.

The anionic substances listed above in (C3) preferably have a certainsize, such as a molecular weight (M) of 1 kDa to 5,000 kDa.

Setting the molecular weight in this range allows the anionic substanceto undergo ionic bonding with the cationic substance and fulfill therole of trapping the cationic substance for an extended period of time.Stable microparticles of an ion complex can thus be formed. Thecytotoxicity of a typical cationic substance, caused by electrostaticinteraction with the membrane surface of a cell, can also be mitigated.

In addition to the anionic substances listed in (C3), it is alsopossible to use a polymer derivative, for example, that substantiallyfunctions as an anionic substance by introducing an anionic functionalgroup into an amino group in the 4-position of poly(4-aminostyrene),which is a cationic polymer, by dehydration synthesis of a dicarboxylicacid such as oxalic acid.

Two or more types of the anionic substances listed above in (C3) may beincluded.

Here, a mixed-type temperature-responsive polymer composition in whichthe ratio ((C2)/(C1)) of (C2) 2-amino-2-hydroxymethyl-1,3-propanediol(tris) to (C1) a polymer of 2-N,N-dimethylaminoethyl methacrylate(DMAEMA) and/or a derivative thereof is 1.0 or less is preferably used.

The ratio ((C2)/(C1)) is designated as the mass ratio.

When using a mixed-type temperature-responsive polymer composition withthe above ratio, the cell structure can be formed more easily in thebelow-described culturing step.

This composition can further improve the balance between hydrophilicityand hydrophobicity of the composition. It is inferred that this suitablebalance favorably adjusts the adhesiveness of cells to the culturingsurface and activates migration and orientation of the cells.

The above ratio ((C2)/(C1)) is preferably 0.1 or greater.

Setting the above ratio to 0.1 or greater facilitates achievement of theaforementioned effect of reducing the cloud point and also facilitatesachievement of the aforementioned effect of easier formation of cellstructures.

For the same reasons as above, the above ratio ((C2)/(C1)) is morepreferably 0.1 to 0.5.

The C/A ratio (positive/negative charge) in the mixed-typetemperature-responsive polymer composition is preferably 0.5 to 16.

In the present disclosure, the C/A ratio refers to the ratio of thepositive charge of substances included in the composition to thenegative charge of substances included in the composition. Specifically,the C/A ratio is represented by the expression ((positive charge perpolymer molecule)×N1)/((negative charge per molecule of anionicsubstance)×N3), where N1 is the number of moles of (C1) the polymer ofDMAEMA and/or a derivative thereof, and N3 is the number of moles of(C3) the anionic substance.

Furthermore, when the anionic substance is DNA in the presentdisclosure, the number of negative charges per molecule of the anionicsubstance is calculated as the number of base pairs (bp number) ofDNA×2, and the molecular weight (Da) is calculated as the bp number×660(the average molecular weight of an AT pair and a CG pair).

Setting the C/A ratio to be 0.5 to 16 facilitates achievement of theaforementioned effect of easier formation of tubular cell structures.

It is inferred that this range makes the balance between negative chargeand positive charge in the composition suitable and can suppresscytotoxicity due to positive charge. It is also inferred that this rangefurther improves the balance between hydrophilicity and hydrophobicityof the composition and can facilitate cell migration and orientation.

For the same reasons as above, the above ratio C/A is more preferably 2to 10 and most preferably near 8.

(Preparation Step)

In an example manufacturing method, a portion of a culturing surface iscoated with the temperature-responsive polymer and/or thetemperature-responsive polymer composition to prepare a coated culturingsurface (preparation step) (see FIG. 1, (i) to (iii)).

Here, the culturing surface apart from the coated culturing surface maybe either cell adhesive or cell non-adhesive, but a cell non-adhesiveculturing surface is preferable to facilitate obtaining a cell mass ofthe desired shape. The production method of the cell non-adhesiveculturing surface is not restricted. For example, a cell culturecontainer provided with a cell non-adhesive culturing surface, such asPrimeSurface® (PrimeSurface is a registered trademark in Japan, othercountries, or both) by SUMILON; a cell culture container not subjectedto surface treatment for cell adhesion, such as a cell culture containerfor culturing Escherichia coli; or a cell non-adhesive sheet, pad, orthe like may be used.

As illustrated in FIG. 1, the coated culturing surface is preferablysurrounded by a non-coated culturing surface to inhibit contact betweencells and the wall and to adjust the shape of the cell mass (see (ii),(iii) of FIG. 1).

The shape of the coated culturing surface may be appropriately adjustedin conjunction with the desired shape of the chondrocyte mass. Examplesinclude a circle, a rectangle, and a donut (ringed) shape in plan view.

The preparation step may, for example, be a step of dissolving atemperature-responsive polymer or a temperature-responsive polymercomposition in a solvent to form a temperature-responsive polymersolution, applying the solution onto the culturing surface of a cellculture container, and drying to prepare a coated cell culture container(preparation step I). The preparation step may also be a step of coolingan aqueous solution including a temperature-responsive polymer or atemperature-responsive polymer composition (temperature-responsivepolymer aqueous solution) to the cloud point of thetemperature-responsive polymer or below, casting the cooledtemperature-responsive polymer aqueous solution onto the culturingsurface of a cell culture container, and heating to a temperature abovethe cloud point to prepare a coated cell culture container (preparationstep II).

Examples of the solvent in the temperature-responsive polymer solutionin preparation step I include water; physiological saline; buffersolutions; alcohols such as methanol, ethanol, n-propyl alcohol,isopropyl alcohol, 1-butanol, isobutyl alcohol, 2-butanol, t-butylalcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol,2,2-dimethyl-1-propanol, 1-pentanol, 2-pentanol, 3-pentanol,2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol,2-methyl-2-pentanol, allyl alcohol, benzyl alcohol, and salicyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, methylpropyl ketone, methyl isobutyl ketone, methyl vinyl ketone,cyclohexanone, 2-methyl cyclopentanone, acetophenone, benzophenone, andisophorone; esters such as methyl acetate, ethyl acetate, propylacetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butylacetate, tert-butyl acetate, vinyl acetate, methyl formate, ethylformate, propyl formate, esters of the aforementioned alcohols andphosphoric acid, and esters of the aforementioned alcohols and carbonicacid; chloroform; benzene; toluene; diethyl ether; and dichloromethane.

Among these, alcohols such as methanol, ethanol, n-propyl alcohol,isopropyl alcohol, 2-butanol, t-butyl alcohol, and allyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, and methylvinyl ketone; esters such as methyl acetate, ethyl acetate, isopropylacetate, tert-butyl acetate, and vinyl acetate; chloroform; benzene;toluene; diethyl ether; and dichloromethane are preferred forfacilitating uniform coating of the culturing surface and for havingexcellent solubility of temperature-responsive polymers. An organicsolvent with a low boiling point (such as at least one selected from thegroup consisting of a low-molecular alcohol with 1 to 4 carbon atoms, alow-molecular ketone with 3 to 5 carbon atoms, and an acetic acid alkylester having an alkyl group with 1 to 4 carbon atoms; in particular, atleast one selected from the group consisting of a low-molecular alcoholwith 1 to 4 carbon atoms, a low-molecular ketone with 3 to 5 carbonatoms, and an acetic acid alkyl ester having an alkyl group with 1 to 4carbon atoms, the low-molecular alcohol, low-molecular ketone, andacetic acid alkyl ester having a boiling point lower than that of water)is more preferable for allowing drying in a short time and facilitatingeven more uniform application on the culturing surface. Methanol andethanol are particularly preferable for their low cost and excellentoperability.

One type of these solvents may be used alone, or a combination of two ormore types may be used.

Since the solvent has excellent solubility with respect to atemperature-responsive polymer, the temperature-responsive polymer tendsnot to become insoluble and precipitate even at a temperature equal toor greater than the cloud point (such as room temperature or 37° C.).This eliminates the need to manage the temperature of thetemperature-responsive polymer during application of thetemperature-responsive polymer, allowing easy preparation of a coatedcell culture container.

In the preparation step I, a hydrophilic molecule is preferably includedin the temperature-responsive polymer to facilitate self-aggregation ofcells. Examples of the hydrophilic molecule include non-ionic,hydrophilic molecules that do not affect the C/A ratio of thetemperature-responsive polymer, such as polyethylene glycol (PEG),dimethyl acrylamide (DMAA), glycerin, Triton X, polypropylene glycol,and the like.

In the preparation step I, the content of the temperature-responsivepolymer in the temperature-responsive polymer solution is preferably0.00075 to 0.015 weight %, more preferably 0.001 to 0.01 weight %,relative to the temperature-responsive polymer solution (100 weight %)to facilitate uniform coating of the culturing surface by thetemperature-responsive polymer.

In the preparation step I, the content of the hydrophilic molecule inthe temperature-responsive polymer solution is preferably 0.00001 to0.00015 weight %, more preferably 0.00003 to 0.0001 weight %, relativeto the temperature-responsive polymer (100 weight %) to facilitateself-aggregation of cells.

To facilitate uniform coating of the culturing surface by thetemperature-responsive polymer or the temperature-responsive polymercomposition, the temperature-responsive polymer solution in thepreparation step I preferably does not include water, and the weightratio of water in the temperature-responsive polymer solution (100weight %) is more preferably 0.5 weight % or less and even morepreferably 0.1 weight % or less.

The weight ratio of water can be measured by a method known to a personskilled in the art, such as gas chromatography or the Karl Fischermethod.

In the preparation step I, the temperature-responsive polymer solutionmay be applied to the entire culturing surface or to a portion of theculturing surface, but application to the entire culturing surface ispreferable to facilitate obtaining a cell structure.

In the preparation step I, preferred conditions for drying the appliedtemperature-responsive polymer solution are drying under atmosphericpressure at a temperature of 10° C. to 70° C. for 1 to 3,000 minutes touniformly coat the culturing surface with the temperature-responsivepolymer or the temperature-responsive polymer composition. Quick dryingof the applied temperature-responsive polymer solution facilitatesuniform coating on the culturing surface with an even distribution ofthe temperature-responsive polymer or the temperature-responsive polymercomposition.

The applied temperature-responsive polymer solution may, for example, bedried by letting the cell culture container stand in an incubator at 37°C.

In the preparation step II, examples of the solvent for dissolving thetemperature-responsive polymer or the temperature-responsive polymercomposition include water; physiological saline; and buffer solutionssuch as a phosphate buffer solution, phosphate buffered saline (PBS),and a tris buffer solution.

In the preparation step II, examples of the method of cooling thetemperature-responsive polymer aqueous solution include placing thetemperature-responsive polymer aqueous solution in a refrigerator atapproximately 4° C. and cooling to a temperature at or below the cloudpoint.

In the preparation step II, examples of the method of casting thetemperature-responsive polymer aqueous solution onto the culturingsurface include a method of tilting the culturing surface of the cellculture container to spread the temperature-responsive polymer aqueoussolution that has a temperature at or below the cloud point and a methodof spreading the temperature-responsive polymer aqueous solution using aspatula.

In the preparation step II, examples of the method of heating the casttemperature-responsive polymer aqueous solution to above the cloud pointinclude a method of letting the cell culture container after the castingstep stand in an incubator at 37° C.

In an example, illustrated in FIG. 1, of a manufacturing method of achondrocyte mass, the preparation step is performed by applying thetemperature-responsive polymer and/or the temperature-responsive polymercomposition to the central portion of the culturing surface of a cellculture container (see (i) of FIG. 1) while drawing a desired shape (see(ii) of FIG. 1) and then drying the applied region (see (iii) of FIG.1).

The preparation step in Embodiment (I) may also be performed by laying amasking sheet (not illustrated) with a hole (cutout) of a desired shapeon the culturing surface of the cell culture container, arranging thetemperature-responsive polymer and/or the temperature-responsive polymercomposition on the sheet, and then removing the sheet.

A material known to a person skilled in the art may be used for themasking sheet, such as a material with a contact angle of 700 or less.Specific examples include polyethylene terephthalate, polystyrene,polycarbonate, glass, polypropylene, and the like modified with ahydrophilic group. In particular, polystyrene or the like withN,N-dimethylacrylylamide introduced and fixed therein by radiation graftpolymerization is preferred to reduce eluates for use in cell culturing.

The shape, size, thickness, and the like of the sheet are notrestricted, but a thickness of 0.05 mm to 2.0 mm is preferred.

The area of the coated culturing surface is not restricted and may, forexample, be 0.5 mm² to 300 mm² when using a ϕ35 mm cell culturecontainer to manufacture a donut-shaped chondrocyte mass with an outerdiameter of 1 mm to 20 mm and an inner diameter of 0.1 mm to 19 mm.

The amount of temperature-responsive polymer and temperature-responsivepolymer composition that the coated culturing surface has per unit areais preferably 0.1 μg/cm² to 3.0 μg/cm² and more preferably 0.5 μg/cm² to2.5 μg/cm².

The zeta potential of the coated culturing surface is preferably 0 mV to50 mV, more preferably 0 mV to 35 mV, and even more preferably 10 mV to25 mV. A zeta potential of 0 mV or more facilitates adhesion ofnegatively charged cells. A zeta potential of 50 mV or less can reducecytotoxicity.

Furthermore, setting the zeta potential in the aforementioned rangesfurther facilitates production of cell structures having an aggregated(pellet-like) shape by simply culturing cells under appropriate cultureconditions. The reason is that setting the surface zeta potential withinthe aforementioned ranges is inferred to provide the coated culturingsurface with a weak positive charge that does not trigger cytotoxicity,to ensure rapid adhesion of the seeded cells, to improve cell activityand encourage secretion of extracellular matrix, and also toappropriately inhibit cell migration, strengthening the bond betweencells.

The zeta potential refers to the value calculated with the Smoluchowskiequation by measurement using a zeta potential meter (for example, model“ELSZ” by Otsuka Electronics Co.) with a particle (zeta potential: −5 mVto +5 mV) in which polystyrene latex is coated with hydroxypropylcellulose as a reference monitor particle.

The contact angle of water relative to the coated culturing surface ispreferably 50° to 90°, more preferably 60° to 80°, and even morepreferably 62° to 78° to increase the effects of Aspect (I).

The contact angle of water relative to the coated culturing surfacerefers to the average contact angle measured in accordance with JISR3257 at any number of points on the coated culturing surface.

FIGS. 3A and 3B are an outline of a modification to the preparationstep, with the subsequent seeding and culturing step.

FIG. 3A illustrates a first modification to the preparation step.

The first modification to the preparation step uses a cell non-adhesivepad that has a planar shape fitting within the culturing surface of thecell culture container, has a predetermined thickness, and has thecentral portion thereof cut out into a desired planar shape (see (i) ofFIG. 3A).

Cell non-adhesive refers to cells not adhering or tending not to adhere.

In the preparation step of the first modification, thetemperature-responsive polymer and/or the temperature-responsive polymercomposition is first arranged on the entire culturing surface of thecell culture container, and the cell non-adhesive sheet is then laid onthe polymer and/or polymer composition. As a result, the coatedculturing surface is surrounded by walls, i.e. the coated culturingsurface is provided on the bottom of the recess.

This first modification allows three-dimensional control of the shape ofthe cell mass during the below-described seeding and culturing step andallows more precise manufacturing of a chondrocyte mass having a desiredshape.

A material known to a person skilled in the art may be used for the cellnon-adhesive pad usable in the first modification, such as a materialwith a contact angle of 70° C. or less. Specific examples includepolyethylene terephthalate, polystyrene, polycarbonate, glass,polypropylene, and the like modified with a hydrophilic group. Inparticular, polystyrene or the like with N,N-dimethylacrylylamideintroduced and fixed therein by radiation graft polymerization ispreferred to reduce eluates for use in cell culturing.

The shape, size, thickness, and the like of the pad are not restricted,but a diameter (maximum diameter) of 0.1 mm to 10 mm is preferable whenusing a ϕ35 mm cell culture container.

In the first modification, a cell non-adhesive pad with a size fittingwithin the culturing surface of the cell culture container and providedwith a recess of a desired planar shape in the central portion thereofmay be used (not illustrated).

In the preparation step in this case, the temperature-responsive polymerand/or the temperature-responsive polymer composition is arranged onlyon the bottom of the recess in the pad (not illustrated). The polymerand/or polymer composition is not arranged on the surface other than thebottom of the recess, i.e. the surface of the walls of the recess andthe pad apart from the recess.

In this example as well, a chondrocyte mass having a desired shape canbe manufactured more precisely in the recess during the below-describedseeding and culturing step.

In Embodiment (I), cell non-adhesive pads of different sizes may beused, for example by allowing a chondrocyte mass to expand in theseeding and culturing step, transferring the chondrocyte mass to therecess of a larger-sized cell non-adhesive pad, and performing the nextseeding and culturing step.

This technique allows the size of the coated culturing surface relativeto the size of the chondrocyte mass to be kept constant in each seedingand culturing step, thus bringing the shape of the chondrocyte masscloser to the desired shape.

FIG. 3B illustrates a second modification to the preparation step.

In the second modification to the preparation step, a recess with adesired planar shape is carved into the culturing surface of the cellculture container (see FIG. 3B).

In the preparation step of the second modification, thetemperature-responsive polymer and/or temperature-responsive polymercomposition is arranged only on the bottom of the carved-out recess (see(i) of FIG. 3B). The polymer and/or polymer composition is not arrangedon the surface other than the bottom of the recess, i.e. the surface ofthe walls of the recess and the sheet apart from the recess.

This second modification allows three-dimensional control of the shapeof the cell mass during the below-described seeding and culturing stepand allows more precise manufacturing of a chondrocyte mass having adesired shape.

In the above-described first and second modifications to the preparationstep, the walls of the recess in particular are preferably cellnon-adhesive to inhibit adhesion between the seeded cells and the wallsof the recess and to adjust the shape of the obtainable cell mass.

(Seeding and Culturing Step)

Next in an example manufacturing method in Embodiment (I), cells capableof differentiating into chondrocytes are seeded onto a coated culturingsurface in the presence of a cell mass, and the cell mass and the cellscapable of differentiating into chondrocytes are cocultured to produce achondrocyte mass (seeding and culturing step).

As illustrated in FIG. 1, after the above-described preparation step andbefore the below-described seeding and culturing step in an examplemanufacturing method of Embodiment (I), cells capable of differentiatinginto chondrocytes are seeded onto the coated culturing surface preparedin the above-described preparation step, and the seeded cells arecultured to produce a cell mass used in the below-described seeding andculturing step (see (iv) to (viii) of FIG. 1).

The manufacturing method of Embodiment (I), however, is not limited tothis example, and the cell mass used in the seeding and culturing stepmay be produced separately (not illustrated), for example in a differentcell culture container.

In the example in FIG. 1, the seeding and culturing step is performed byproviding a cell mass on the coated culturing surface, adding cells anda cell culture medium to the cell culture container (see (i) of FIG. 2),subsequently placing the cell culture container in a typical 37° C. cellincubator (see (ii) of FIG. 2), adding new cell culture medium by mediumexchange (see (iii) of FIG. 2), and placing the cell culture containeragain in the cell incubator (see (iv) and (v) of FIG. 2). Theparentheses in (iv) of FIG. 2 contain a cross-sectional view of thestructure.

In this step, the cell mass is preferably present in the central portionof the coated culturing surface, as illustrated in (i) of FIG. 2, toadjust the overall shape of the chondrocyte mass.

The cell culture medium used before medium exchange and the cell culturemedium used after medium exchange may be selected appropriately inaccordance with purpose or use. For example, the medium before exchangemay be a growth medium, and the medium after exchange may be adifferentiation medium or a redifferentiation medium.

Examples of the cells capable of differentiating into chondrocytesinclude chondrocytes, adipose, synovium, fascia, periosteum, periodontalmembrane, dental pulp, mesenchymal stem cells derived from bone marrow,and iPS cells.

One type of these cells capable of differentiating into chondrocytes maybe used alone, or a combination of two or more types may be used.

The cell density when seeding cells in the seeding and culturing step is0.3×10⁴ cells/cm² or more, preferably 0.3×10⁵ cells/cm² or more, andmore preferably 0.5×10⁵ cells/cm² or more. Furthermore, to preventproblems related to the cell cycle, such as growth arrest due to contactbetween cells during culturing, the cell density is preferably 10.0×10⁵cells/cm² or less and more preferably 4.5×10⁵ cells/cm² or less.

A person skilled in the art can appropriately determine the cultureconditions on the basis of the type of cells being used and the purposeof the experiment. Example conditions are 37° C. and a 5% CO₂atmosphere.

The phenomenon that occurs in the seeding and culturing step isdescribed below with reference to FIG. 2.

In this step, the seeded cells first precipitate onto the coatedculturing surface, on which a cell mass is present in the centralportion, and onto the non-coated culturing surface. At this time, thecells that precipitate on the coated culturing surface adhere to thecoated culturing surface and survive, whereas the cells that precipitateon the non-coated culturing surface survive without adhering to thenon-coated culturing surface (see (ii) of FIG. 2). The cells that do notadhere, however, are removed by suction during the first medium exchangeafter seeding (see (iii) of FIG. 2). These cells are preferably removedrapidly to inhibit the release of harmful components, such as heat shockproteins and inflammatory cytokines that accompany apoptosis.

Upon further culturing of the cells adhered to the coated culturingsurface, the cells located near the boundary between the coatedculturing surface and the non-coated culturing surface start toaggregate from the coated culturing surface, along with the cellspositioned closer to the central portion of the coated culturingsurface, so as to surround the cell mass in the central portion (see(iv) of FIG. 2). In other words, the cells that were adhered peel offtowards the central portion of the coated culturing surface, separatingfrom the culturing surface, so that periphery of the entire sheet ofcells warps. Ultimately, the cell mass provided in advance and theseeded cells integrate to form a layered structure in a cross-sectionalview (see (v) of FIG. 2).

The cells, used in the seeding and culturing step of Aspect (I), capableof differentiating into cartilage pass through the aforementionedaggregation process, differentiate into chondrocytes, and mature. As isknown by a person skilled in the art, however, chondrocytes are cellswith a special property allowing a hypoxic state upon maturing.Accordingly, the chondrocytes in the cell mass obtained through theaggregation process can survive even in a hypoxic state, and thesechondrocytes can continue to survive even when surrounded by new cellscapable of differentiating into cartilage and placed in a hypoxic statein the next seeding and culturing step.

Apart from the coated culturing surface, the seeded cells alsoprecipitate on the cell mass, and these cells are integrated togetherduring cell aggregation.

In an example manufacturing method in Embodiment (I), theabove-described seeding and culturing step is performed multiple times(see (vi) of FIG. 2).

A large chondrocyte mass can be obtained by performing the seeding andculturing step multiple times, and the size can be adjusted inaccordance with purpose and use.

This embodiment allows a cell mass constituted by mature cells formed ina previous seeding and culturing step to be successively surrounded byimmature cells in subsequent seeding and culturing steps, thereby makingmaturation of cells capable of differentiating into cartilage compatiblewith growth of a cell mass.

Immature cells (cells that grow but have insufficient cartilageproperties) and mature cells (cells that do not grow but have acquiredthe properties of chondrocytes, such as the characteristics of survivalin a hypoxic environment and highly elastic flexibility) can becontrolled by appropriately selecting the medium. For example, mixing adifferentiation inducing factor such as TGF-β1 into the medium that isused can encourage cell differentiation.

The length of time from when the cell mass is formed in the centralportion of the coated culturing surface in the previous seeding andculturing step until cells are seeded in the next seeding and culturingstep is not restricted. This length of time may be set appropriately bya person skilled in the art in view of maturing the formed cell massinto a chondrocyte mass and inhibiting a reduction in activity of thechondrocyte mass, and comprehensively considering the type,concentration, and the like of the redifferentiation medium being used.

(Chondrocyte Mass)

The chondrocyte mass of Embodiment (I) is manufactured by themanufacturing method of a chondrocyte mass of Embodiment (I).

The size of the chondrocyte mass is not restricted. The diameter(maximum diameter) may be 1 mm to 100 mm, and particularly in the caseof a donut-shaped chondrocyte mass, the outer diameter may be 3 mm to 50mm, and the inner diameter may be 0.3 mm to 49 mm.

(Manufacturing Method of Graft Material)

The manufacturing method of a graft material of Embodiment (I) includesthe step of seeding mesenchymal cells in the presence of the chondrocytemass manufactured with the manufacturing method of a chondrocyte mass ofEmbodiment (I) and coculturing the chondrocyte mass and the mesenchymalcells to produce a graft material.

FIG. 4 is an overview of the manufacturing method of a graft material ofEmbodiment (I).

Apart from using mesenchymal cells, the aforementioned step in themanufacturing method of a graft material in Embodiment 1 may beperformed in the same way as the above-described seeding and culturingstep in the manufacturing method of a chondrocyte mass of Embodiment(I).

Examples of mesenchymal cells include chondrocytes, fibroblasts, andADSC.

(Graft Material) The graft material of Embodiment (I) is manufactured bythe manufacturing method of a graft material of Embodiment (I).

Mesenchymal cells, in particular fibroblasts, are present on theoutermost side of a chondrocyte mass in the graft material of Embodiment(I). The graft material therefore tends to adhere firmly to the tissuesurrounding the graft site during grafting onto a living organism. Thiscan increase the curative effects at the graft site and yield a betterprognosis.

FIG. 5 is an overview of another example manufacturing method of achondrocyte mass in Embodiment (I).

The other example manufacturing method of a chondrocyte mass inEmbodiment (I) includes the above-described first modification to thepreparation step.

Specifically, the other example manufacturing method uses a cellnon-adhesive pad (with a thickness of 1 mm, for example) provided at thecentral portion with a cutout that is donut-shaped in plan view (forexample, outer diameter (ϕo) of 8 mm, inner diameter (ϕi) of 4 mm, andwidth of 2 mm).

Here, the outer contour line of the coated culturing surface and theinner contour line of the coated culturing surface preferably formconcentric circles to improve the donut shape of the chondrocyte mass.

The details of each step in the other example manufacturing method of achondrocyte mass in Embodiment (I) may be similar to the steps in theabove-described example manufacturing method of the chondrocyte mass inEmbodiment (I) (see (i) to (ix) of FIG. 5).

When using a ϕ35 mm plate as the cell culture container, for example, inthe other example manufacturing method, the width of the coatedculturing surface is preferably 3 mm or less, more preferably 2.5 mm orless, and the height of the wall is preferably 3 mm or less, morepreferably 2.5 mm or less, to obtain a chondrocyte mass with a betterdonut shape (ringed shape).

(Manufacturing Method of Composite Material)

A manufacturing method of a composite material of Embodiment (I)includes a composite body production step of fitting a donut-shapedchondrocyte mass in particular, among the chondrocyte masses ofEmbodiment (I), onto a tubular structure to produce a composite body anda culturing step of culturing the composite body to produce a compositematerial.

The manufacturing method of a composite material of Embodiment (I)preferably includes a step of preparing a tubular structure and a corematerial (see (i) of FIG. 6) and then inserting the core material into ahollow portion of the tubular structure from one end to the other (see(ii) of FIG. 6), a composite body production step of fitting adonut-shaped chondrocyte mass of Embodiment (I) onto the tubularstructure to produce a composite body (see (iii) of FIG. 6), a culturingstep of culturing the composite body to produce a composite material(see (iv) of FIG. 6), and a step of removing the core material from thecomposite material (see (v) of FIG. 6).

FIG. 6 is an overview, in (i) to (v), of an example manufacturing methodof a composite material in Embodiment (I).

Details of each step in an example manufacturing method of a chondrocytemass in Embodiment (I) are provided below.

The tubular structure may have a hollow portion and may be a biotube(synthetic blood vessel), a collagen tube, an elastin tube, apolygluconic acid tube, a polylactic acid tube, or the like.

A biotube may have collagen as the principal component and may, forexample, be produced with the method disclosed in the Examples ofJP2004261260A.

The outer diameter of the tubular structure may be similar to the innerdiameter of the donut-shaped cell structure of Embodiment (I), such as 1mm to 100 mm. The inner diameter of the tubular structure is notrestricted and may, for example, be 0.1 mm to 50 mm. The length of thetubular structure may be appropriately set in accordance with thepurpose or use and is not restricted. The length may, for example, befrom 1 mm to 300 mm.

A solid body or a porous body may be used as the core material. Specificexamples include a silicon rod-like structure, an acrylic rod-likestructure, a metallic rod-like structure, bisque, and a metal meshcompressed structure. When using a porous body, medium and oxygen canalso expand from the luminal side of the composite body.

The outer diameter of the core material may be similar to the innerdiameter of the tubular structure, such as 0.1 mm to 50 mm.

The core material may be similar to the tubular structure in length ormay be longer or shorter than the tubular structure by, for example, 0.1mm to 10 mm.

The composite body production step may, for example, be performed bypassing the tubular structure (with the core material provided on theinside) produced in the above-described step through the circle of thedonut-shaped chondrocyte mass of Embodiment (I) while securing thechondrocyte mass in place with tweezers.

The number of donut-shaped chondrocyte masses per tubular structure maybe determined appropriately in accordance with purpose or use and is notrestricted. The number may, for example, be from 1 to 1,000.

When using a plurality of chondrocyte masses, the distance betweenadjacent chondrocyte masses may be set appropriately in accordance withpurpose or use and is not restricted. The distance may, for example, befrom 0.1 mm to 100 mm before the below-described subsequent culturingstep.

The time from when the tubular structure is completely fitted in theabove-described composite body production step until culturing of thecomposite body starts in the below-described culturing step ispreferably 1 min to 180 min, more preferably 1 min to 120 min, tomaintain cell activity.

The culturing step may, for example, be performed by placing thecomposite body produced in the above-described composite body productionstep under appropriate conditions (such as 37° C., 5% CO₂ atmosphere)for a predetermined length of time (such as from 12 hours to 150 days).

The culturing step can also be performed inside a living organism, forexample by embedding the above-described composite body inside theliving organism and leaving the composite body inside for apredetermined length of time to allow the composite body to integratewith biological tissue forming around the outer surface of the compositebody.

The core material can be removed from the composite materialappropriately by using tweezers or the like.

The operations in the above step are not restricted and may be performedby hand or by using machines or apparatuses.

(Composite Material)

In the composite material of Embodiment (I), donut-shaped chondrocytemasses in particular among the chondrocyte masses of Embodiment (I) areprovided on the outer surface of a tubular structure (see (v) of FIG.6).

The material, outer diameter, inner diameter, length, and the like ofthe tubular structure, along with the number of donut-shaped chondrocytemasses per tubular structure, may be as described above in themanufacturing method of a composite material of Embodiment (I).

When using a plurality of chondrocyte masses, the distance betweenadjacent chondrocyte masses (cartilage tissue) may be determinedappropriately in accordance with purpose or use and is not restricted.For example, the distance may be from 0.1 mm to 100 mm, or adjacentchondrocyte masses (cartilage tissue) may overlap (i.e. a distance of 0mm).

In the composite material of Embodiment (I), a core material may beprovided inside the tubular structure (see (v) of FIG. 6).

The material, outer diameter, length, and the like of the core materialmay be as described above in the manufacturing method of a compositematerial of Embodiment (I).

The composite material of Embodiment (I) may be manufactured with themanufacturing method of a composite material of the present embodiment.

The chondrocyte mass, graft material, and composite material ofEmbodiment (I) are useful for treatment of joints, the trachea, thenose, and the like and more specifically are useable in treatment of ameniscus, tracheal cartilage, nasal cartilage, ear cartilage,intervertebral discs, articular cartilage, ligaments, the Achillestendon, and the like.

For example, the manufacturing method of a chondrocyte mass, graftmaterial, and composite material of Embodiment (I) allows a chondrocytemass and graft material to be shaped in accordance with a CAD drawingbased on a CT image of the affected area of a patient. The manufacturingmethod of a chondrocyte mass, graft material, and composite material ofthe present embodiment therefore has the potential of contributinggreatly to achieving customized medical treatment.

[[Aspect (II)]]

Epithelial cells do not adhere easily to a cell culture container.Insufficient adhesion to the culture container leads to problems such asan unstable cell form after growth, making stable cell culturingdifficult with known methods. Furthermore, actin filaments areundeveloped in epithelial cells, and the binding between cells is weak.Weak adhesion to the cell culture container then tends to cause theepithelial cells to peel from the cell culturing surface. A spheroidformed from epithelial cells also tends not to adhere to a cell culturecontainer but rather to float in the cell culture container andtherefore has an extremely high risk of accidentally being suctionedduring medium exchange or the like.

With regard to Aspect (II), we discovered that a cell culture containercoated with a temperature-responsive polymer has excellent adhesivenessfor epithelial cells. In particular, we discovered that cells other thanepithelial cells adhere to the cell culture container coated with atemperature-responsive polymer with an appropriate force and often havethe property of self-aggregation when the density reaches a certainlevel, whereas when placing epithelial cells in a cell culture containercoated with a temperature-responsive polymer, the epithelial cellsadhere strongly to the culturing surface, unlike other cells. We alsodiscovered that using a cell culture container coated with atemperature-responsive polymer facilitates culturing of epithelial cellsand achieves an excellent cell form during growth. Furthermore, wediscovered a method allowing efficient formation of a cell structure ofepithelial cells in which the cell structure tends not to peel from thecoated cell culturing surface and tends not to be accidentally suctionedduring medium exchange or the like.

[Culture Method of Epithelial Cells]A culture method of epithelial cellsof Aspect (II) includes a production step of producing atemperature-responsive polymer or a temperature-responsive polymercomposition, a culture container preparation step of forming a coatedregion A by coating at least a portion of a culturing surface of a cellculture container with the temperature-responsive polymer or thetemperature-responsive polymer composition to prepare a coated cellculture container including the coated region A, a seeding step ofseeding epithelial cells in the coated cell culture container, and aculturing step of culturing the epithelial cells adhered to the coatedregion A. The concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition in the coated region A is 0.3pg/mm² or more.

The culture method of epithelial cells of Aspect (II) preventsepithelial cells from unintentionally peeling during culturing, allowingepithelial cells to be cultured easily.

The entire culturing surface of the coated cell culture container may bethe coated region A, or a portion of the culturing surface may be thecoated region A. The culturing surface may have one coated region A or aplurality of coated regions A.

(Production Step)

Examples of the production step in Embodiment (II) include a stepsimilar to the production step in Aspect (I), and a similar step ispreferred.

The temperature-responsive polymer and temperature-responsive polymercomposition used in the culture method in Embodiment (II) are preferably(A) for having better adhesiveness of the epithelial cells.

The production step of producing a mixture containing2-N,N-dimethylaminoethyl methacrylate (DMAEMA) in Embodiment (II) isalso referred to as a mixture production step.

(Culture Container Preparation Step)

In the culture method of an embodiment of Aspect (II) (Embodiment (II)),the culture container preparation step is a step of forming a coatedregion A by coating at least a portion of a culturing surface of a cellculture container with the temperature-responsive polymer or thetemperature-responsive polymer composition to prepare a coated cellculture container including the coated region A.

The culture container preparation step may, for example, be a step ofdissolving a temperature-responsive polymer or a temperature-responsivepolymer composition in a solvent to form a temperature-responsivepolymer solution, applying the solution onto the culturing surface of acell culture container, and drying to prepare a coated cell culturecontainer (culture container preparation step I). The culture containerpreparation step may instead be a step of cooling an aqueous solutionincluding a temperature-responsive polymer or a temperature-responsivepolymer composition (temperature-responsive polymer aqueous solution) tothe cloud point of the temperature-responsive polymer or below, castingthe cooled temperature-responsive polymer aqueous solution onto theculturing surface of a cell culture container, and heating to atemperature above the cloud point to prepare a coated cell culturecontainer (culture container preparation step II).

Here, the temperature-responsive polymer solution used when forming thecoated region A is also referred to as temperature-responsive polymersolution A, and the temperature-responsive polymer aqueous solution usedwhen forming the coated region A is also referred to astemperature-responsive polymer aqueous solution A.

Examples of the solvent in the temperature-responsive polymer solutionin the culture container preparation step I include water; physiologicalsaline; buffer solutions; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 1-butanol, isobutyl alcohol, 2-butanol,t-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol,2,2-dimethyl-1-propanol, 1-pentanol, 2-pentanol, 3-pentanol,2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol,2-methyl-2-pentanol, allyl alcohol, benzyl alcohol, and salicyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, methylpropyl ketone, methyl isobutyl ketone, methyl vinyl ketone,cyclohexanone, 2-methyl cyclopentanone, acetophenone, benzophenone, andisophorone; esters such as methyl acetate, ethyl acetate, propylacetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butylacetate, tert-butyl acetate, vinyl acetate, methyl formate, ethylformate, propyl formate, esters of the aforementioned alcohols andphosphoric acid, and esters of the aforementioned alcohols and carbonicacid; chloroform; benzene; toluene; diethyl ether; and dichloromethane.

Among these, water; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 2-butanol, t-butyl alcohol, and allylalcohol; ketones such as acetone, ethyl methyl ketone, diethyl ketone,and methyl vinyl ketone; esters such as methyl acetate, ethyl acetate,isopropyl acetate, tert-butyl acetate, and vinyl acetate; chloroform;benzene; toluene; diethyl ether; and dichloromethane are preferred forfacilitating uniform coating of the culturing surface and for havingexcellent solubility of temperature-responsive polymers. An organicsolvent with a low boiling point (such as at least one selected from thegroup consisting of a low-molecular alcohol with 1 to 4 carbon atoms, alow-molecular ketone with 3 to 5 carbon atoms, and an acetic acid alkylester having an alkyl group with 1 to 4 carbon atoms; in particular, atleast one selected from the group consisting of a low-molecular alcoholwith 1 to 4 carbon atoms, a low-molecular ketone with 3 to 5 carbonatoms, and an acetic acid alkyl ester having an alkyl group with 1 to 4carbon atoms, the low-molecular alcohol, low-molecular ketone, andacetic acid alkyl ester having a boiling point lower than that of water)is more preferable for allowing drying in a short time and facilitatingeven more uniform application on the culturing surface. Methanol andethanol are particularly preferable for their low cost and excellentoperability.

One type of these solvents may be used alone, or a combination of two ormore types may be used.

Since the solvent has excellent solubility with respect to atemperature-responsive polymer, the temperature-responsive polymer tendsnot to become insoluble and precipitate even at a temperature equal toor greater than the cloud point (such as room temperature or 37° C.).This eliminates the need to manage the temperature of thetemperature-responsive polymer during application of thetemperature-responsive polymer, allowing easy preparation of a coatedcell culture container.

In the culture container preparation step I, a hydrophilic molecule maybe added to the temperature-responsive polymer solution as appropriateto adjust the adhesiveness of the epithelial cells. Examples of thehydrophilic molecule include non-ionic, hydrophilic molecules that donot affect the C/A ratio of the temperature-responsive polymer, such aspolyethylene glycol (PEG), dimethyl acrylamide (DMAA), glycerin, TritonX, polypropylene glycol, and the like.

In the culture container preparation step I, the content of thetemperature-responsive polymer in the temperature-responsive polymersolution A is preferably 0.00000009 mass % to 0.01 mass %, morepreferably 0.0000009 mass % to 0.01 mass %, relative to thetemperature-responsive polymer solution (100 mass %) to facilitateuniform coating of the culturing surface by the temperature-responsivepolymer.

In the culture container preparation step I, the content of thehydrophilic molecule in the temperature-responsive polymer solution A ispreferably 0.00001 mass % to 0.00015 mass %, more preferably 0.00003mass % to 0.0001 mass %, relative to the temperature-responsive polymer(100 mass %) to facilitate self-aggregation of cells.

In the culture container preparation step I, the temperature-responsivepolymer solution A may be applied to the entire culturing surface or toa portion of the culturing surface.

When the temperature-responsive polymer solution is applied to a portionof the culturing surface, one coated region or a plurality of coatedregions may be provided on the culturing surface. A cell culturecontainer with a cell non-adhesive culturing surface is preferably usedwhen the temperature-responsive polymer solution is applied to a portionof the culturing surface.

The temperature-responsive polymer solution may be applied to have auniform concentration over the entire coated region A or applied thicklyto one portion and thinly to other portions.

“Cell non-adhesive” refers to adherent cells (for example, fibroblasts,cardiomyocytes, vascular endothelial cells, and the like) either notadhering or tending not to adhere under normal culture conditions. “Cellnon-adhesive” thus also includes the case of “low-adhesive”.

In the culture container preparation step I, preferred conditions fordrying the applied temperature-responsive polymer solution are dryingunder atmospheric pressure at a temperature of 10° C. to 70° C. for 1 to3,000 minutes to uniformly coat the culturing surface with thetemperature-responsive polymer or the temperature-responsive polymercomposition. Quick drying of the applied temperature-responsive polymersolution facilitates uniform coating on the culturing surface with aneven distribution of the temperature-responsive polymer or thetemperature-responsive polymer composition.

The applied temperature-responsive polymer solution may, for example, bedried by letting the cell culture container stand in an incubator at 37°C.

In the culture container preparation step II, examples of the solventfor dissolving the temperature-responsive polymer or thetemperature-responsive polymer composition include water; physiologicalsaline; and buffer solutions such as a phosphate buffer solution,phosphate buffered saline (PBS), and a tris buffer solution.

In the culture container preparation step II, examples of the method ofcooling the temperature-responsive polymer aqueous solution includeplacing the temperature-responsive polymer aqueous solution in arefrigerator at approximately 4° C. and cooling to a temperature at orbelow the cloud point.

In the culture container preparation step II, examples of the method ofcasting the temperature-responsive polymer aqueous solution onto theculturing surface include a method of tilting the culturing surface ofthe cell culture container to spread the temperature-responsive polymeraqueous solution that has a temperature at or below the cloud point anda method of spreading the temperature-responsive polymer aqueoussolution using a spatula.

In the culture container preparation step II, examples of the method ofheating the cast temperature-responsive polymer aqueous solution toabove the cloud point include a method of letting the cell culturecontainer after the casting step stand in an incubator at 37° C.

Examples of the cell culture container include commercially availablemultiwell plates, dishes, flasks, and the like. Examples of the materialof the cell culture container include polystyrene, polyethyleneterephthalate (PET), polypropylene, polybutene, polyethylene,polycarbonate, and glass. Among these, polystyrene and polyethyleneterephthalate (PET) are preferable for being easy to mold precisely, forallowing adoption of various sterilization methods, and for beingsuitable for microscope observation by virtue of being transparent.

Cell adhesion treatment or the like may be applied to the culturingsurface of the cell culture container, or the surface may be untreated.The culturing surface may be coated, processed, or the like to adjustthe cell adhesiveness.

The planar shape of the culturing surface is not restricted and may, forexample, be a substantially rectangular shape or other substantiallypolygonal shape, a substantially circular shape, or the like. Amongthese, a substantially circular shape is preferred to facilitateobtaining a more homogeneous cell structure.

The bottom shape of the culturing surface (the cross-sectional shape ofthe bottom) is not restricted, and examples include a flat bottom, roundbottom (U-bottom), V-bottom, and uneven bottom. In particular, in theculture method of epithelial cells and the below-described culturingstep of a cell structure, the epithelial cells are a spheroidal cellstructure.

At least a portion of the culturing surface may have a depression. Inthis case, the depression is preferably provided within the coatedregion A and is preferably located in the central portion of the coatedregion A.

The depth of the depression is, for example, preferably 0.001 mm to 10.0mm and more preferably 0.01 mm to 1 mm.

The area of the depression in plan view is, for example, preferably 0.01mm² to 10.0 mm² and more preferably 0.1 mm² to 1 mm².

The area of the culturing surface of the cell culture container ispreferably 150 cm² or less, more preferably 21 cm² or less, and evenmore preferably 200 mm² or less. The lower limit on the area of theculturing surface of the cell culture container is not restricted, andany commercially available size may be used.

The area of the coated region A is preferably 150 cm² or less, morepreferably 21 cm² or less, and even more preferably 200 mm² or less.

The area of the coated region A relative to the entire area (100%) ofthe culturing surface of the cell culture container is preferably from50% to 100% and more preferably from 80% to 100%.

The amount of temperature-responsive polymer per unit area in the coatedregion A is 0.3 pg/mm² or more, preferably 3.0 pg/mm² or more, morepreferably 30 pg/mm² or more, and preferably 200 ng/mm² or less. Theseranges allow the epithelial cells to adhere to the culturing surface andfacilitate culturing.

The zeta potential of the coated region A in the coated cell culturecontainer is preferably 0 mV to 50 mV, more preferably 0 mV to 35 mV,and even more preferably 10 mV to 25 mV. A zeta potential of 0 mV ormore facilitates adhesion of negatively charged cells. A zeta potentialof 50 mV or less can reduce cytotoxicity.

Furthermore, setting the zeta potential in the aforementioned rangesfurther improves the adhesiveness between epithelial cells and thecoated region A. The reason is that setting the surface zeta potentialwithin the aforementioned ranges is inferred to provide the coatedregion A with a weak positive charge that does not trigger cytotoxicity,to ensure rapid adhesion of the seeded cells, to improve cell activityand encourage secretion of extracellular matrix, and also toappropriately inhibit cell migration, strengthening the bond betweencells.

The zeta potential refers to the value calculated with the Smoluchowskiequation by measurement using a zeta potential meter (for example, model“ELSZ” by Otsuka Electronics Co.) with a particle (zeta potential: −5 mVto +5 mV) in which polystyrene latex is coated with hydroxypropylcellulose as a reference monitor particle.

The contact angle of water relative to the coated region A is preferably50° to 90°, more preferably 60° to 80°, and even more preferably 620 to780 to increase the effects of Aspect (II). The contact angle of waterrelative to the coated region A refers to the average contact anglemeasured in accordance with JIS R3257 at any number of points on thecoated region A.

(Seeding Step)

The seeding step is a step of seeding epithelial cells on the coatedcell culture container. Cells may be seeded a portion at a time.

Examples of epithelial cells include cultured cells derived from livercancer, such as HepG2, HepaRG, and HepaRA frequently used in drugdiscovery tests; hepatocytes; cells derived from pancreatic cancer suchas BxPC-3; and primary cultured cells of these types collected from aliving organism. Among these, cultured cells derived from liver cancer,such as HepaRG and HepaRA, in which all of the inherent cell propertiesare average and which are well known to a person skilled in the art, arepreferable for drug discovery tests. Primary cells are suitable foranticancer drug development and laboratory testing.

One type of the aforementioned epithelial cells may be used alone, or acombination of two or more types may be used.

In the seeding step, cells other than the aforementioned epithelialcells may be included, such as mesenchymal stem cells or stromal cells.

In the seeding step, the density of seeded epithelial cells on thecoated region A is not restricted, as long as the density is not so lowthat the epithelial cells die out. For example, a confluency relative tothe surface area of the coated region A of 5% to 100% is preferable, aconfluency of 50% to 100% is more preferable, and a confluency of 80% to100% is even more preferable. Epithelial cells can more easily becultured if the seeded cell density is within the aforementioned ranges.

The density of seeded cells on the coated region A is not restricted, aslong as the density is not so low that the epithelial cells die out. Thedensity is preferably 20 cells/mm² to 15,000 cells/mm², for example, andmore preferably 50 cells/mm² to 1,500 cells/mm². For example, whenseeding by adding 25 μL of cell fluid to a 384 well cell culture platewith a culturing surface area of 8.4 mm², the number of cells in thecell fluid is preferably 7 cells/μL to 5040 cells/μL. Live cells areseeded.

The coated cell culture container may be left to stand in an incubatorat 37° C. and subsequently removed and placed on a clean bench at roomtemperature, for example, with cell seeding then being performed.

Cells are preferably seeded after being diluted in a medium. The mediumfor dilution may be any medium in which epithelial cells can becultured.

(Culturing Step)

The culturing step is a step of culturing the epithelial cells adheredto the coated region A.

The epithelial cells may be cultured by, for example, using a typicalcell incubator at 37° C.

The cultured epithelial cells can be washed with PBS or the like, thenpeeled off using trypsin, trypsin-EDTA, commercially available cellpeeling solution, or the like, and subsequently diluted and passaged.

[Epithelial-Mesenchymal Transition Inducer]

We discovered that the temperature-responsive polymer andtemperature-responsive polymer composition used in the above-describedculture method of an embodiment of Aspect (II) are effective forinducing transition of epithelial cells to mesenchymal cells, i.e.epithelial-mesenchymal transition (EMT).

In other words, an epithelial-mesenchymal transition inducer of Aspect(II) includes the temperature-responsive polymer andtemperature-responsive polymer composition used in the above-describedculture method of an embodiment of Aspect (II) and is preferablycomposed of the temperature-responsive polymer andtemperature-responsive polymer composition.

Known techniques for inducing epithelial-mesenchymal transition includetechniques for inducing EMT using growth factors such as transforminggrowth factor (TGF)-β, epidermal growth factor (EGF), and the like, andtechniques for inducing EMT by culturing epithelial cancer cells on typeIV collagen.

However, when using substances derived from natural products as thegrowth factor, such as TGF-β or EGF, in the above-described knowntechniques, unknown substances or pathogenic substances might beincluded in the growth factor. Variation between lots might alsoadversely affect the reproducibility of experiments. Use of a growthfactor produced with a genetic modification technique also leads toproblems such as endotoxins derived from Escherichia coli contaminatingthe growth factor, or the resulting protein undergoing host-specificEscherichia coli glycosylation and having a structure and propertiesdifferent from growth factors derived from natural products.

While the mechanism is unclear, the epithelial-mesenchymal transitioninducer of Aspect (II) allows induction of mesenchymal transition bysimply culturing epithelial cells on a culturing surface coated with a100% chemically synthesized temperature-responsive polymer andtemperature-responsive polymer composition, without requiring a growthfactor. This yields a low risk of contamination by substances that couldadversely affect transition and yields low variation between lots. Thestrict temperature control required when using a growth factor is alsounnecessary, and animal resources can also be conserved. Theepithelial-mesenchymal transition inducer of Aspect (II) caninexpensively and simply achieve epithelial-mesenchymal transition.

Examples of the temperature-responsive polymer andtemperature-responsive polymer composition in Embodiment (II) include(A) a temperature-responsive polymer containing 2-N,N-dimethylaminoethylmethacrylate (DMAEMA) units and anionic monomer units, (B) atemperature-responsive polymer containing N-isopropyl acrylamide (NIPAM)units, cationic monomer units, and anionic monomer units, and (C) atemperature-responsive polymer composition containing a polymer of2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and/or a derivativethereof, 2-amino-2-hydroxymethyl-1,3-propanediol (tris), and one or moreanionic substances selected from the group consisting of nucleic acids,heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid,polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfatedpolysaccharide, curdlan, polyarginic acid, and alkali metal saltsthereof, as described above in Embodiment (II). Among these, (A) ispreferred for having better adhesiveness of the epithelial cells.

Examples of (A) include (A-1) a temperature-responsive polymer obtainedby a method of polymerizing DMAEMA in the presence of water and (A-2) atemperature-responsive polymer containing a polymer block principallycontaining DMAEMA (polymer chain α terminal) and a copolymer blockprincipally containing DMAEMA and an anionic monomer (polymer chain ωterminal). Of these, (A-1) is preferred for better induction ofepithelial-mesenchymal transition.

The details of the temperature-responsive polymer andtemperature-responsive polymer composition of (A) to (C) may be asdescribed above.

In Embodiment (II), the effect of epithelial-mesenchymal transition byan epithelial-mesenchymal transition inducer can be achieved by forminga coated region A by coating at least a portion of a culturing surfaceof a cell culture container with a temperature-responsive polymer ortemperature-responsive polymer composition to prepare a coated cellculture container including the coated region A, next seeding epithelialcells in the coated cell culture container, and subsequently culturingthe epithelial cells adhered to the coated region A.

The details of the culture container preparation, cell seeding, and cellculturing are not restricted and may be similar to those of the culturemethod of epithelial cells of Embodiment (II) and the manufacturingmethod of a cell structure of Embodiment (II).

[Manufacturing Method of a Cell Structure]

A manufacturing method of a cell structure of Aspect (II) includes aproduction step of producing a temperature-responsive polymer or atemperature-responsive polymer composition, a culture containerpreparation step of forming a coated region A by coating at least aportion of a culturing surface of a cell culture container with thetemperature-responsive polymer or the temperature-responsive polymercomposition to prepare a coated cell culture container including thecoated region A, a seeding step of seeding epithelial cells in thecoated cell culture container, and a culturing step of forming anaggregated cell structure from the epithelial cells to obtain a cellstructure adhered to the coated region A. The concentration of thetemperature-responsive polymer or the temperature-responsive polymercomposition in the coated region A is 0.3 pg/mm² or more.

(Production Step)

Examples of the production step in the manufacturing method of a cellstructure of Embodiment (II) include a step similar to the productionstep in the above-described culture method of epithelial cells ofEmbodiment (II).

(Culture Container Preparation Step)

Examples of the production step in the manufacturing method of a cellstructure of Embodiment (II) include a step similar to the productionstep in the above-described culture method of epithelial cells ofEmbodiment (II).

In the culture container preparation step I of the production step inthe manufacturing method of a cell structure of Embodiment (II), thecontent of the temperature-responsive polymer in thetemperature-responsive polymer solution A is preferably 0.00000009 mass% to 0.0001 mass %, more preferably 0.00000009 mass % to 0.0000009 mass%, relative to the temperature-responsive polymer solution (100 mass %)to facilitate uniform coating of the culturing surface by thetemperature-responsive polymer.

The area of the culturing surface of the cell culture container in theproduction step of the manufacturing method of a cell structure ofEmbodiment (II) is preferably 200 mm² or less, more preferably 50 mm² orless, and even more preferably 10 mm² or less, to further facilitatemanufacturing of a cell structure that includes epithelial cells. Thelower limit on the area of the culturing surface of the cell culturecontainer is not restricted, and any commercially available size may beused.

The area of the coated region A in the production step of themanufacturing method of a cell structure of Embodiment (II) ispreferably 10 mm² or less, more preferably 1.0 mm² or less, and evenmore preferably 0.1 mm² or less, so that the cell structure includingepithelial cells adheres more firmly to the coated region A and so thatthe cell structure tends not to peel due to operations such aspipetting.

The area the coated region A relative to the entire area (100%) of theculturing surface of the cell culture container in the production stepof the manufacturing method of a cell structure of Embodiment (II) ispreferably from 0.1% to 50% and more preferably from 0.1% to 10%.

To facilitate formation of a cell structure, the coated region Apreferably not only covers the bottom of the cell culture container butalso reaches the side surface.

The amount of temperature-responsive polymer per unit area in the coatedregion A in the production step of the manufacturing method of a cellstructure of Embodiment (II) is 0.3 pg/mm² or more, preferably 0.3pg/mm² to 200 pg/mm², more preferably 0.3 pg/mm² to 150 pg/mm², and evenmore preferably 0.3 pg/mm² to 9 pg/mm². These ranges allow the cellstructure to adhere to the coated region A more firmly.

In the production step of the manufacturing method of a cell structureof Embodiment (II), the temperature-responsive polymer or thetemperature-responsive polymer composition is preferably applied to theentire culturing surface to obtain a cell structure with abundantsecretion of extracellular matrix and high cell activity.

Cell culturing operations are often performed with an automaticincubator when culturing cells in a 384-well plate or a 1536-well plate,for which operations such as medium exchange are difficult to performmanually, or when culturing cells in numerous wells. During operationswith an automatic incubator, however, it is difficult to adjust theposition of the suction port that suctions the medium in each well toavoid accidentally suctioning suspended cell structures. An automaticincubator thus tends to accidentally suction suspended cell structures,yielding a low culturing efficiency of cell structures. In particular,cell structures of epithelial cells tend not to adhere to the culturingsurface, causing frequent accidental suctioning.

If the concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition is high at the bottom of theculturing surface (for example, the coated region A) and is low or zeroon the side surface, the epithelial cells temporarily adhere to the sidesurface but tend to peel because of gravity. The epithelial cells thatpeel off from the side surface and fall onto the bottom assemble to forma cell structure, which adheres firmly to the bottom. Hence, a cellstructure of epithelial cells that is easy to produce and is not proneto accidental suction during medium exchange can be formed. Furthermore,if the concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition on the side surface is low,the epithelial cells adhere to the side surface once and secrete cellmatrix but then peel off and fall to the bottom. A cell structure thatincludes epithelial cells with high cell activity can thus be obtained.

Examples of a cell culture container in which the concentration of atemperature-responsive polymer or a temperature-responsive polymercomposition is high on the bottom of the culturing surface and low onthe side surface include a cell culture container having a coated regionA on at least a portion of the culturing surface and a coated region Bon at least a portion of the culturing surface at a different positionthan the coated region A. The coated region B is preferably formed tosurround the coated region A. For example, by providing the coatedregion A at the central portion (deepest portion) of a culturing surfacewith a round bottom and providing the coated region B in the portionother than the coated region A, the epithelial cells on the coatedregion B fall due to gravity and gather in the coated region A in thecentral portion to more easily form a cell structure. The formed cellstructure adheres firmly to the coated region A and thus tends not topeel off due to operations such as pipetting.

The concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition in the coated region B ispreferably lower than in the coated region A, is more preferably lessthan 200 pg/mm², even more preferably less than 100 pg/mm², and stillmore preferably 50 pg/mm² or less.

The concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition in the coated region B ispreferably 5% to 90%, more preferably 10% to 50%, of the concentrationof the temperature-responsive polymer or the temperature-responsivepolymer composition in the coated region A.

The concentration of the temperature-responsive polymer or thetemperature-responsive polymer composition in the coated region B may beuniform throughout the region or higher in one portion and lower inother portions. The coated region B may also include a portion withoutthe temperature-responsive polymer or the temperature-responsive polymercomposition.

In the production step of the manufacturing method of a cell structureof Embodiment (II), the bottom of the culturing surface (thecross-sectional shape of the bottom) is preferably a round bottom(U-bottom), V-bottom, or uneven bottom to facilitate gathering of theepithelial cells in the deepest portion of the culturing surface forformation of a cell structure. A round bottom (U-bottom, spindle bottom)is particularly preferable to facilitate formation of a spheroidal cellstructure.

In a cell culture container having a round bottom (U-bottom, spindlebottom), the radius of curvature R of the contour line of the bottom ofthe culturing surface in a cross-section along the depth direction ofthe cell culture container is, on average over the entire round bottom(U-bottom, spindle bottom), preferably 50 mm or less, more preferably 10mm or less, even more preferably 5 mm or less, and particularlypreferably 2 mm or less, and preferably 0.1 mm or more, more preferably0.2 mm or more, even more preferably 0.4 mm or more, and particularlypreferably 0.8 mm or more.

The maximum width L of the cell culture container is preferably 100 mmor less, more preferably 50 mm or less, even more preferably 20 mm orless, and particularly preferably 10 mm or less, and preferably 1 mm ormore, more preferably 2 mm or more, even more preferably 3 mm or more,and particularly preferably 4 mm or more.

The deepest portion of the round bottom (U-bottom, spindle bottom) isnot limited to the shape illustrated in FIG. 15 and may have apredetermined width in plan view.

Examples of a method of setting the concentration of thetemperature-responsive polymer or the temperature-responsive polymercomposition to be high in the coated region A and to be lower in thecoated region B than in the coated region A include using a culturecontainer provided with a depression at the bottom of the culturingsurface (see (i) of FIG. 16), adding a temperature-responsive polymersolution, and drying while the amount of temperature-responsive polymersolution is larger in the depression and smaller on the side surface.

Examples of the shape of the depression are listed above.

(Seeding Step)

Examples of the seeding step in the manufacturing method of a cellstructure of Embodiment (II) include a step similar to the seeding stepin the above-described culture method of epithelial cells of Embodiment(II).

In the seeding step in the manufacturing method of a cell structure ofEmbodiment (II), the density of seeded epithelial cells on the coatedregion A is not restricted, as long as the density is not so low thatthe epithelial cells die out. For example, a confluency relative to thesurface area of the coated region A of 5% to 100% is preferable, aconfluency of 50% to 100% is more preferable, and a confluency of 80% to100% is even more preferable. A cell structure of epithelial cells canmore easily be produced if the seeded cell density is within theaforementioned ranges.

In the seeding step, the number of seeded cells is preferably 20cells/mm² or more, more preferably 50 cells/mm² or more, even morepreferably 100 cells/mm² or more, and particularly preferably 500cells/mm² or more, and preferably 15,000 cells/mm² or less, 10,000cells/mm² or less, 5,000 cells/mm² or less, or 1,500 cells/mm² or lessto facilitate formation of a cell structure. For example, when seedingby adding 25 μL of cell fluid to a 384 well cell culture plate with aculturing surface area of 8.4 mm², the number of cells in the cell fluidis preferably 7 cells/μL to 5040 cells/μL. Live cells are seeded.

(Culturing Step)

The culturing step is a step of forming an aggregated cell structurefrom the epithelial cells to obtain a cell structure adhered to thecoated region A.

The seeded cells are preferably left to stand. The temperature at whichthe seeded cells are left to stand is not restricted. For example, atemperature of 30° C. or more is preferable, 35° C. to 38° C. is morepreferable, and 37° C. is even more preferable.

The time for which the seeded cells are left to stand is not restricted.For example, the cells are preferably cultured for 1 hour to 240 hoursand more preferably for 10 hours to 96 hours.

An example manufacturing method of a cell structure of Embodiment (II)is described below with reference to FIGS. 15 and 16.

FIG. 15 illustrates an example manufacturing method of a cell structurethat includes epithelial cells.

A temperature-responsive polymer or a temperature-responsive polymercomposition is applied to coat a cell culture container having a roundculturing surface (for example, a 384-well plate or 1536-well plate),thereby preparing a coated cell culture container including a coatedregion A (see (i), (ii) of FIG. 15). Subsequently, a cell fluid dilutedwith medium and including epithelial cells is added to the coated cellculture container (see (iii) of FIG. 15). The seeded epithelial cellsadhere to the entire coated region A (see (iv) of FIG. 15). In thisexample, the density of the epithelial cells is 100% confluency (see(iv) of FIG. 15). By adhering once to the coated region A, theepithelial cells can secrete an extracellular matrix component andmaintain high cell activity. The epithelial cells adhered to the sidesurface in the coated region A peel off from the coated region A becauseof gravity and gather at the bottom of the culturing surface. Theepithelial cells gathered at the bottom adhere to form a cell structure(see (v) of FIG. 15). The formed cell structure is adhered to the coatedcell culture container at the culturing surface bottom (see (v) of FIG.15).

In this example, the concentration of the temperature-responsive polymeror the temperature-responsive polymer composition in the coated region Ais such that the epithelial cells adhered to the side surface peel offbecause of gravity, whereas the epithelial cells adhered to the bottomadhere strongly enough for the cells to tend not to peel off due tooperations such as pipetting.

FIG. 16 illustrates an example manufacturing method of a cell structurethat includes epithelial cells.

A solution containing a temperature-responsive polymer or atemperature-responsive polymer composition is added to a cell culturecontainer with a round-bottom culturing surface having a depression(recess) on the bottom (for example, a 384-well plate or 1536-wellplate). The solution gathers in the depression on the bottom, and thecoated region A with a high concentration of the temperature-responsivepolymer or the temperature-responsive polymer composition forms in thedepression. On the other hand, the coated region B with a lowerconcentration of the temperature-responsive polymer or thetemperature-responsive polymer composition than the coated region Aforms on the side surface, because the amount of solution on the sidesurface is less than in the depression. A coated cell culture containerincluding the coated region A and the coated region B is thus prepared(see (i) and (ii) of FIG. 16). In the coated region B on the sidesurface, epithelial cells adhere temporarily but then tend to peelbecause of gravity. The epithelial cells that fall after peeling offfrom the side surface assemble to form a cell structure that firmlyadheres to the coated region A on the bottom. This helps to avoidaccidental suction during medium exchange (see (ii) of FIG. 16). A cellfluid diluted with medium and including epithelial cells is added to thecoated cell culture container (see (iii) of FIG. 16). The seededepithelial cells adhere to the coated region A and the coated region B(see (iv) of FIG. 16). In this example, the density of the epithelialcells is 100% confluency (see (iv) of FIG. 16). By adhering once to thecoated region A and the coated region B, the epithelial cells cansecrete an extracellular matrix component and maintain high cellactivity. The epithelial cells adhered to the side surface in the coatedregion B peel off from the coated region B because of gravity and gatherat the bottom of the culturing surface. The epithelial cells gathered atthe bottom adhere and form a cell structure (see (v) of FIG. 16). Theformed cell structure is adhered to the coated cell culture container atthe coated region A (see (v) of FIG. 16).

[Cell Culture Container for Epithelial Cells]

A cell culture container for epithelial cells of Aspect (II) includes acoated region A, coated with a temperature-responsive polymer or atemperature-responsive polymer composition, on at least a portion of theculturing surface. The concentration of the temperature-responsivepolymer or the temperature-responsive polymer composition in the coatedregion A is 0.3 pg/mm² or more.

Epithelial cells do not easily peel off during culturing with the cellculture container for epithelial cells of Aspect (II), allowingepithelial cells to be cultured easily. A cell structure containingepithelial cells can also be manufactured easily.

Examples of the temperature-responsive polymer and thetemperature-responsive polymer composition in the cell culture containerfor epithelial cells of Embodiment (II) include the above-describedexamples.

Examples of the cell culture container for epithelial cells ofEmbodiment (II) include the above-described coated cell culturecontainer. The cell culture container for epithelial cells of Embodiment(II) may, for example, be manufactured by the above-described productionstep and culture container preparation step.

[[Aspect (III)]]

[Production Apparatus of Three-Dimensional Tissue Body]

A production apparatus of a three-dimensional tissue body of Embodiment(III) includes a culturing surface having one or more through holes, ashaft inserted through the one or more through holes, and one or morecoated culturing surfaces where the culturing surface is coated by atemperature-responsive polymer or a temperature-responsive polymercomposition. At least one of the one or more through holes is locatedwithin one of the one or more coated culturing surfaces, and theculturing surface is movable in an extending direction of the shaft.

In Aspect (III), the “cloud point” of the polymer does not necessaryhave the strict meaning of “a predetermined temperature such that thepolymer dissolves below the temperature but becomes insoluble andprecipitates at or above the temperature”, but also refers to “apredetermined temperature such that below the temperature, 10 minutes ormore are required to dissolve a polymer that has become insoluble andprecipitated”.

The production apparatus of a three-dimensional tissue body ofEmbodiment (III) has few components, is relatively easy to use as amedical product, and has a low risk of infection. The productionapparatus can also be placed inside a sealed plastic container toproduce a three-dimensional tissue body hygienically and aseptically. Byhaving few components and being small, the production apparatus can alsoreduce the amount of waste when disposed of as a medical product.

(Shaft)

The shaft is not restricted, as long as the dimensions of the resultingthree-dimensional tissue body are stable. Examples of the material forthe shaft include plastics such as polystyrene, polyethyleneterephthalate (PET), polypropylene, polybutene, polyethylene, acrylicresin, polyurethane resin, urea resin, and polycarbonate; rubberelastomers such as silicon rubber, chloroprene rubber (such asNeoprene®), and SBR; ceramic; glass; and metallic/inorganic materialsuch as stainless steel, titanium, and Nitinol® (Neoprene and Nitinolare registered trademarks in Japan, other countries, or both). Amongthese, plastics such as polystyrene, polyurethane resin, acrylic resin,and polycarbonate; and metals such as stainless steel and Nitinol® arepreferred for allowing application of various sterilization methods,producing little eluate, and having a track record as a medicalmaterial.

The surface of the shaft may be cell adhesive or cell non-adhesive. Thesurface of the shaft is preferably cell non-adhesive, however, tofacilitate retaining the structure of the three-dimensional tissue bodywound around the shaft. Proteins are preferably not included on thesurface of the shaft to facilitate peeling (removal) from the shaft of athree-dimensional tissue body including substances secreted from cells.

The cell adhesiveness of the shaft surface can be adjusted by a methodof coating the shaft surface with a cell adhesive substance, a method ofcovering the shaft surface with a film of cell adhesive substance, amethod of performing radiation/plasma discharge to introduce a celladhesive molecular group into the shaft surface, and the like.

Examples of the cell adhesive substance include laminin, collagen,fibronectin, peptides, cationic polymers, and polystyrene. Examples ofthe peptides include peptides containing an arginine-glycine-asparticacid sequence and peptides containing a sequence of 8 or moreconsecutive arginine residual groups. Examples of the cationic polymersinclude aminostyrene. Among these, laminin, collagen, and fibronectin,which have high cell adhesiveness, are preferable. Reagents containingthe above-listed cell adhesive substances can also be suitably used.Examples of such reagents include serum.

The ringed three-dimensional tissue body that winds around the shaft iscovered by an extracellular matrix secreted by the tissue body itself.Hence, the three-dimensional tissue body can be wound around the shaftwithout performing a process to make the surface of the shaft celladhesive.

“Cell adhesive” refers to adherent cells (for example, vascularendothelial cells, vascular cells, chondrocytes, fibroblasts, and thelike) adhering under normal culture conditions. “Cell adhesive” thusalso includes the case of “low-adhesive”.

The three-dimensional tissue body with a ringed shape, a luminal shape,or the like formed to be wound around the shaft can be peeled off theshaft surface even when the shaft surface is cell adhesive. When theshaft surface is cell non-adhesive, such as Teflon® (Teflon is aregistered trademark in Japan, other countries, or both), siliconrubber, or a hydrophilic coating, then by covering the shaft surfacewith a collagen tube, for example, the three-dimensional tissue bodywith a ringed shape, a luminal shape, or the like can be removedtogether with the collagen tube.

Examples of the cross-sectional shape of the shaft in a planeperpendicular to the extending direction (length direction) of the shaftinclude a substantially circular shape, a substantially polygonal shape,a half-moon shape, a crescent shape, a string shape, and a tear shape.Among these, a substantially circular shape is preferred to obtain athree-dimensional tissue body close to the shape of a cartilaginousring, a blood vessel, the trachea, or the like. In other words, theshaft preferably has a substantially cylindrical shape.

The cross-sectional shape of the shaft may be the same or differentalong the extending direction in a plane perpendicular to the extendingdirection.

The shape of the shaft in the extending direction may be linear (seeFIGS. 18 to 20) or curved, such as a C-shape, a U-shape, or a spiral.

The dimensions of the shaft are not restricted. For example, the lengthof the shaft is preferably 0.1 mm to 600 mm and more preferably 1 mm to300 mm. When the cross-sectional shape of the shaft in a planeperpendicular to the extending direction is substantially circular, themaximum diameter thereof is preferably 0.01 mm to 150 mm and morepreferably 0.1 mm to 50 mm.

The shaft surface may be smooth or uneven. The surface may include ahole or may be a mesh or a porous surface with multiple pores.

The shaft may also be hollow.

Among these options, the shaft is preferably hollow, and the shaftsurface is preferably porous, because this configuration can maintainhigh cell activity by stable providing medium components and oxygen toall of the cells included in the three-dimensional tissue body woundaround the shaft and can rapidly eliminate cell metabolites.

When the shaft is a porous material constituted by a sponge-likecontinuous foam, cells might infiltrate into the shaft, and cells thatinfiltrate deeply might die out, or the formed three-dimensional tissuebody and the shaft might adhere stubbornly, making it difficult to peeloff the three-dimensional tissue body. The maximum hole diameter on theshaft surface is therefore preferably 200 μm or less. When the shaft isconstituted by a fiber assembly, such as a woven fabric of metallicfiber, the cells adhere easily to the shaft surface, and the fiberassembly might affect cell differentiation. The maximum hole diameter istherefore preferably 10 μm or more in this case.

When a plurality of shafts are used, the material, dimensions, shape,and the like of each shaft may be the same or different. The distancebetween shafts is not restricted, and shafts may be in contact with eachother, for example.

(Culturing Surface)

At least a portion of the culturing surface includes at least one coatedculturing surface that is coated by a temperature-responsive polymer ora temperature-responsive polymer composition. One coated culturingsurface or a plurality of coated culturing surfaces may be provided onthe culturing surface.

When one coated culturing surface is provided, the entire culturingsurface may be the coated culturing surface (see FIGS. 18, 20), or aportion may be the coated culturing surface (see FIG. 19). Between theseoptions, the entire culturing surface is preferably the coated culturingsurface for greater manufacturing ease.

When the culturing surface is double sided, such as a plate or disc,either one side or both sides may be the coated culturing surface.

Examples of the material of the culturing surface include polystyrene,polyethylene terephthalate (PET), polypropylene, polybutene,polyethylene, polycarbonate, glass, silicon resin, acrylic resin, andpolyurethane resin. Among these, polystyrene, polyethyleneterephthalate, glass, silicon resin, and acrylic resin are preferablefor being easy to mold precisely and for allowing adoption of varioussterilization methods.

Cell adhesion treatment or the like may be applied to the culturingsurface, or the surface may be untreated. The culturing surface may becoated, processed, or the like to adjust the cell adhesiveness.

The planar shape of the culturing surface is not restricted and may, forexample, be a substantially polygonal shape (a substantially polygonalshape having a through hole) such as a substantially rectangular shape,a substantially circular shape (a substantially circular shape having athrough hole, such as a ring), or the like.

The area of the culturing surface is preferably 0.1 mm² to 150 cm², morepreferably 8.4 mm² to 21 cm², to further facilitate manufacturing of athree-dimensional tissue body with a ringed shape, a luminal shape, orthe like.

The bottom shape of the culturing surface (the cross-sectional shape ofthe bottom) is not restricted, and examples include a flat bottom, roundbottom, and uneven bottom. Among these, a flat bottom is preferable tofacilitate obtaining a three-dimensional tissue body with a ringedshape, a luminal shape, or the like.

A plurality (for example, 2 or more, 5 or more, 10 or more, or the like)of culturing surfaces may be provided to allow simultaneous formation ofa plurality of three-dimensional tissue bodies and to allow moreefficient formation of three-dimensional tissue bodies with a luminalshape in a shorter time. No upper limit is placed on the number ofculturing surfaces, as long as the number is in a range allowingseeding, culturing, and the like of cells and allowing production ofthree-dimensional tissue bodies.

When a plurality of culturing surfaces are provided, one shaft ispreferably inserted through the through hole of at least two of theculturing surfaces, and one shaft is more preferably inserted throughthe through hole of all of the culturing surfaces.

The production apparatus of a three-dimensional tissue body ofEmbodiment (III) may, for example, have one or a plurality of members inwhich a shaft is inserted through a plurality of culturing surfaces(i.e. a member constituted by a shaft and a plurality of culturingsurfaces, with the culturing surfaces disposed like shelves in theextending direction of the shaft).

When a plurality of culturing surfaces are provided, the distancesbetween culturing surfaces in the extending direction of the shaft maybe the same or different. For example, the distance between culturingsurfaces in the extending direction of the shaft is preferably 10 timesor less and more preferably 7.5 times or less the length, in theextending direction of the shaft, of the cells to be used to facilitateconnection between ringed or luminal three-dimensional tissue bodiesproduced on the culturing surfaces. Specifically, the distance betweenculturing surfaces in the extending direction of the shaft is preferably0.1 mm to 10 mm, more preferably 0.2 mm to 2.0 mm.

Connection of ringed or luminal three-dimensional tissue bodies producedon the culturing surfaces includes not only adhesion between cellsconstituting the ringed or luminal three-dimensional tissue bodiesproduced on the culturing surfaces, but also connection between ringedor luminal three-dimensional tissue bodies, produced on the culturingsurfaces, via proteins secreted by cells constituting the ringed orluminal three-dimensional tissue bodies produced on the culturingsurfaces (such as proteins constituting the extracellular matrix).

Examples of the temperature-responsive polymer or thetemperature-responsive polymer composition of the coated culturingsurface include those listed below in the production step.

The content of the temperature-responsive polymer or thetemperature-responsive polymer composition per unit area of the coatedculturing surface is preferably 5 ng/mm² to 50 ng/mm², more preferably15 to 40 ng/mm², to facilitate obtaining a three-dimensional tissue bodywith a ringed shape, a luminal shape, or the like.

When a plurality of coated culturing surfaces are provided, the contentof the temperature-responsive polymer or the temperature-responsivepolymer composition included per unit area of the coated culturingsurfaces may be the same or different on the coated culturing surfaces.

The planar shape of the coated culturing surface is not restricted andmay, for example, be a substantially polygonal shape (a substantiallypolygonal shape having a through hole) such as a substantiallyrectangular shape, a substantially circular shape (a substantiallycircular shape having a through hole, such as a ring), or the like.Among these, a substantially circular shape is preferred to facilitateobtaining a three-dimensional tissue body with a more homogeneousdistribution of cells.

When a plurality of coated culturing surfaces are provided, the planarshape of the coated culturing surfaces may be the same or different.

The surface area of the coated culturing surface is preferably 0.1 mm²to 150 cm², more preferably 8.4 mm² to 21 cm², to facilitate obtaining athree-dimensional tissue body with a more homogeneous distribution ofcells. When a plurality of coated culturing surfaces are provided, thesurface areas of the coated culturing surfaces may be the same ordifferent.

If the area of the coated culturing surface is small, the number ofcells wound around the shaft decreases, facilitating formation of athree-dimensional tissue body with cell matrix as the principalcomponent. The area of the coated culturing surface when forming athree-dimensional tissue body with cell matrix as the principalcomponent may, for example, be 0.1 mm² to 50 mm².

The surface area of the coated culturing surface can be measured with amethod well known to a person skilled in the art, such as image analysisof a photomicrograph.

The zeta potential of the coated culturing surface is preferably 0 mV to50 mV, more preferably 0 mV to 35 mV, and even more preferably 10 mV to25 mV. A zeta potential of 0 mV or more facilitates adhesion ofnegatively charged cells. A zeta potential of 50 mV or less can reducecytotoxicity.

Furthermore, setting the zeta potential in the aforementioned rangesallows the seeded cells to be formed into a ringed three-dimensionaltissue body wound around a shaft by simply culturing cells underappropriate culture conditions. The reason is that setting the surfacezeta potential within the aforementioned ranges is inferred to providethe coated culturing surface with a weak positive charge that does nottrigger cytotoxicity, to ensure rapid adhesion of the seeded cells, toimprove cell activity and encourage secretion of extracellular matrix,and also to appropriately inhibit cell migration, strengthening the bondbetween cells.

When a plurality of coated culturing surfaces are provided, the zetapotential of the coated culturing surfaces may be the same or different.

The zeta potential refers to the value calculated with the Smoluchowskiequation by measurement using a zeta potential meter (for example, model“ELSZ” by Otsuka Electronics Co.) with a particle (zeta potential: −5 mVto +5 mV) in which polystyrene latex is coated with hydroxypropylcellulose as a reference monitor particle.

The contact angle of water relative to the coated culturing surface ispreferably 50° to 90°, more preferably 60° to 80°, and even morepreferably 620 to 780 to increase the effects of Aspect (III). When aplurality of coated culturing surfaces are provided, the contact angleof water relative to each coated culturing surface may be the same ordifferent.

The contact angle of water relative to the coated culturing surfacerefers to the average contact angle measured in accordance with JISR3257 at any number of points on the coated culturing surface.

The culturing surface includes at least one through hole. When onethrough hole is included, the through hole is preferably in the centralportion of the culturing surface. Insertion of the shaft through thethrough hole yields a production apparatus of a three-dimensional tissuebody in which the culturing surface and the shaft are integrated.

The planar shape of the through hole is not restricted and may, forexample, be a substantially polygonal shape or a substantially circularshape. The planar shape of the through hole is preferably the same asthe cross-sectional shape of the shaft in a plane perpendicular to theextending direction of the shaft to facilitate obtaining athree-dimensional tissue body with a more uniform distribution of cells.Among these options, the cross-sectional shape of the shaft in a planeperpendicular to the extending direction of the shaft and the planarshape of the through hole are preferably both substantially circular(see FIGS. 18 to 20).

As long as the shaft can be inserted through the through hole, the shapeof the through hole may be the same as or different from thecross-sectional shape of the shaft in a plane perpendicular to theextending direction of the shaft. When a plurality of through holes areprovided, the planar shape of each through hole may be the same ordifferent.

At least one of the through holes is provided within the coatedculturing surface, and preferably only one is provided.

The through hole is provided within the coated culturing surface, onethrough hole is preferably provided in the central portion of the coatedculturing surface, and the one through hole is more preferably providedin a portion including the center of mass of the coated culturingsurface. When the coated culturing surface is substantially circular,the through hole is preferably provided in the region within 0.75 r fromthe center of the coated culturing surface (where r is the radius of thecoated culturing surface). If the through hole is provided in thecentral portion of the coated culturing surface, the direction in whichcells aggregate can be concentrated towards the central portion,allowing production of a three-dimensional cell body with a morehomogeneous distribution of cells. Shifting the through hole from thecentral portion of the coated culturing surface allows production of athree-dimensional tissue body in which the thickness of the ring is notuniform.

One through hole is preferably provided within one coated culturingsurface to facilitate obtaining a three-dimensional tissue body with agood dimensional shape.

When a plurality of coated culturing surfaces are provided in theproduction apparatus of Embodiment (III), it suffices to provide atleast one through hole within at least one coated culturing surfaceamong all of the coated culturing surfaces. The number of coatedculturing surfaces and the number of through holes may be equal, withone through hole provided within each coated culturing surface. Forexample, the production apparatus of Embodiment (III) may be aproduction apparatus that includes five coated culturing surfaces andone through hole, with five coated culturing surfaces provided on theculturing surface and the one through hole provided within one of thecoated culturing surfaces; a production apparatus that includes fivecoated culturing surfaces and five through holes, with five coatedculturing surfaces provided on the culturing surface and one throughhole provided within each coated culturing surface; or the like.

The surface area of the through hole is preferably 0.1 mm² to 150 cm²,more preferably 8.4 mm² to 21 cm². When the through hole issubstantially circular, the maximum diameter is preferably 0.01 mm to150 mm. When a plurality of through holes are provided, the surface areaof each through hole may be the same or different.

On a coated culturing surface provided with one through hole within thecoated culturing surface, the ratio of the surface area of the throughhole to the surface area of the coated culturing surface (100%) ispreferably 0.1% to 50%, more preferably 1% to 30%. A ratio within theaforementioned ranges further facilitates obtaining a three-dimensionaltissue body wound around the shaft.

(Positional Relationship Between Shaft and Culturing Surface) Itsuffices for the shaft to be inserted through the through hole. Theshaft and culturing surface may be in contact (see FIGS. 18, 19), or agap may be provided between the shaft and culturing surface (see FIG.20). The gap between the shaft and the culturing surface is preferably adistance allowing aggregated cells to jump over the gap and wind aroundthe shaft and/or a distance at which a three-dimensional tissue bodywound around the shaft does not peel off when the culturing surface ismoved in the extending direction of the shaft. The gap is morepreferably 5.0 mm or less and even more preferably 0.5 mm or less.

When a plurality of the culturing surfaces are provided, a gap ispreferably provided between the shaft and the culturing surface tofacilitate a uniform distribution of cells on each culturing surface andto facilitate connection of the ringed or luminal three-dimensionaltissue bodies produced on the culturing surfaces.

The culturing surface is movable in the extending direction of the shaftand is preferably moveable in the direction from the coated culturingsurface toward the culturing surface to achieve a low risk of thethree-dimensional tissue body wound around the shaft suffering physicalstress due to medium flow produced when the culturing surface is movedand to facilitate obtaining a three-dimensional tissue body with aringed shape, a luminal shape, or the like. The angle between theculturing surface and the extending direction of the shaft is notrestricted, but a right angle is preferable.

When a plurality of through holes are provided, a shaft may be insertedthrough all of the through holes or only a portion of the through holes.A shaft is preferably inserted through all of the through holes toprevent the seeded cells from falling below the culturing surface and toallow the seeded cells to adhere to the coated culturing surface.

An example of a production apparatus of a three-dimensional tissue bodyof Embodiment (III) is described below with reference to FIGS. 18 to 21and FIG. 26. The production apparatuses in FIGS. 18 to 21 and FIG. 26 ispreferable for having few components, being relatively easy to use as amedical product, having a low risk of infection, and allowing easyproduction of biotubes. Among these apparatuses, the productionapparatuses of a three-dimensional tissue body in FIG. 20 and FIG. 26are preferable to further facilitate obtaining a three-dimensionaltissue body with a ringed shape, a luminal shape, or the like.

FIG. 18 is an outline of an example production apparatus of athree-dimensional tissue body of Embodiment (III).

In this example, a coated culturing surface coated by atemperature-responsive polymer or a temperature-responsive polymercomposition is provided on one entire side of a disc-shaped culturingsurface. The coated culturing surface has a through hole in the centralportion, and a cylindrical shaft is inserted through the through hole.The coated culturing surface and the shaft are in contact, with no gaptherebetween. The culturing surface is perpendicular to the extendingdirection of the shaft and is movable in the extending direction of theshaft. The culturing surface in FIGS. 18 to 21 is movable both in theupward direction (from the culturing surface towards the coatedculturing surface) and downward direction (from the coated culturingsurface towards the culturing surface) of the figures. The direction inwhich the culturing surface moves, the movement distance, and/or themovement timing may, for example, be controlled by a computer or thelike automatically.

FIG. 19 is an outline of an example production apparatus of athree-dimensional tissue body of Embodiment (III).

In this example, a coated culturing surface coated by atemperature-responsive polymer or a temperature-responsive polymercomposition is provided on a portion of one side of a disc-shapedculturing surface.

FIG. 20 is an outline of an example production apparatus of athree-dimensional tissue body of Embodiment (III).

In this example, a coated culturing surface coated by atemperature-responsive polymer or a temperature-responsive polymercomposition is provided on one entire side of a disc-shaped culturingsurface. A gap is provided between the coated culturing surface and theshaft. In other words, the diameter of the shaft is less than the holediameter of the through hole.

FIG. 21 is a photograph of an example production apparatus of athree-dimensional tissue body of Embodiment (III).

In this example, a coated culturing surface coated by atemperature-responsive polymer is provided on one entire side of adisc-shaped, plastic culturing surface. The coated culturing surface hasa through hole in the central portion, and a cylindrical shaft isinserted through the through hole. The shaft is hollow, and the surfaceof the shaft is a mesh. A metal rod is inserted inside the shaft tosecure the production apparatus. A gap is provided between the coatedculturing surface and the shaft. The culturing surface is perpendicularto the extending direction of the shaft and is movable in the extendingdirection of the shaft.

The production apparatus of a three-dimensional tissue body ofEmbodiment (III) is placed inside a container with a slightly largerinner diameter than the outer diameter of the culturing surface. As aresult, cells seeded from the upper portion of FIG. 21 tend not to fallbelow the culturing surface, and the seeded cells can be caused toadhere to the coated culturing surface.

FIG. 26 is an outline of an example production apparatus of athree-dimensional tissue body of Embodiment (III).

In this example, two disc-shaped culturing surfaces are provided, and acoated culturing surface coated by a temperature-responsive polymer or atemperature-responsive polymer composition is provided on one entireside of each culturing surface. One shaft is inserted through thethrough holes of the two culturing surfaces, and ringed or luminalthree-dimensional tissue bodies can be formed simultaneously on the twoculturing surfaces. A gap is provided between each coated culturingsurface and the shaft. By adjustment of the distance between the twoculturing surfaces, the ringed or luminal three-dimensional tissuebodies produced on the two culturing surfaces can easily be connected byadhesion between cells included in the three-dimensional tissue bodiesor via proteins secreted by the cells included in the three-dimensionaltissue bodies.

(Manufacturing Method of Production Apparatus of Three-DimensionalTissue Body)

Examples of manufacturing methods of a production apparatus of athree-dimensional tissue body of Embodiment (III) include a methodincluding a production step of producing a temperature-responsivepolymer or a temperature-responsive polymer composition and a coatedculturing surface preparation step of coating the culturing surface withthe temperature-responsive polymer or the temperature-responsive polymercomposition to prepare a coated culturing surface.

—Production Step—

Examples of the production step in Embodiment (III) include a stepsimilar to the production step in Aspect (I), and a similar step ispreferred.

The temperature-responsive polymer and temperature-responsive polymercomposition used in the culture method in Embodiment (III) arepreferably (A) to further facilitate obtaining a three-dimensionaltissue body with a ringed shape, a luminal shape, or the like.

The production step of producing a mixture containing2-N,N-dimethylaminoethyl methacrylate (DMAEMA) in Embodiment (III) isalso referred to as a mixture production step.

In Embodiment (III), the temperature-responsive polymer of (A-1) allowsformation of a three-dimensional tissue body with a ringed shape, aluminal shape, or the like by culturing cells under appropriate cultureconditions, as described below.

In Embodiment (III), the temperature-responsive polymer of (A-2) allowsformation of a three-dimensional tissue body with a ringed shape, aluminal shape, or the like by culturing cells under appropriate cultureconditions, as described below.

In Embodiment (III), the temperature-responsive polymer of (B) allowsformation of a three-dimensional tissue body with a ringed shape, aluminal shape, or the like by culturing cells under appropriate cultureconditions, as described below.

In Embodiment (III), use of a mixed-type temperature-responsive polymercomposition with the above ratio facilitates formation of athree-dimensional tissue body in the below-described culturing step.

Setting the C/A ratio to be 0.5 to 16 in Embodiment (III) facilitatesachievement of the aforementioned effect of easier formation of athree-dimensional tissue body with a ringed shape, a luminal shape, orthe like.

—Coated Culturing Surface Preparation Step—

The coated culturing surface preparation step is a step of coating aculturing surface with the temperature-responsive polymer or thetemperature-responsive polymer composition to prepare a coated culturingsurface.

The coated culturing surface preparation step may, for example, be astep of dissolving a temperature-responsive polymer or atemperature-responsive polymer composition in a solvent to form atemperature-responsive polymer solution, applying the solution onto aculturing surface, and drying to prepare a coated culturing surface(coated culturing surface preparation step I), or a step of cooling anaqueous solution including a temperature-responsive polymer or atemperature-responsive polymer composition (temperature-responsivepolymer aqueous solution) to the cloud point of thetemperature-responsive polymer or below, casting the cooledtemperature-responsive polymer aqueous solution onto a culturingsurface, and heating to a temperature above the cloud point to prepare acoated culturing surface (coated culturing surface preparation step II).

Examples of the solvent in the temperature-responsive polymer solutionin the coated culturing surface preparation step I include water;physiological saline; buffer solutions; alcohols such as methanol,ethanol, n-propyl alcohol, isopropyl alcohol, 1-butanol, isobutylalcohol, 2-butanol, t-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol,2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-pentanol, 2-pentanol,3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol,2-methyl-2-pentanol, allyl alcohol, benzyl alcohol, and salicyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, methylpropyl ketone, methyl isobutyl ketone, methyl vinyl ketone,cyclohexanone, 2-methyl cyclopentanone, acetophenone, benzophenone, andisophorone; esters such as methyl acetate, ethyl acetate, propylacetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butylacetate, tert-butyl acetate, vinyl acetate, methyl formate, ethylformate, propyl formate, esters of the aforementioned alcohols andphosphoric acid, and esters of the aforementioned alcohols and carbonicacid; chloroform; benzene; toluene; diethyl ether; and dichloromethane.

Among these, water; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 2-butanol, t-butyl alcohol, and allylalcohol; ketones such as acetone, ethyl methyl ketone, diethyl ketone,and methyl vinyl ketone; esters such as methyl acetate, ethyl acetate,isopropyl acetate, tert-butyl acetate, and vinyl acetate; chloroform;benzene; toluene; diethyl ether; and dichloromethane are preferred forfacilitating uniform coating of the culturing surface and for havingexcellent solubility of temperature-responsive polymers. An organicsolvent with a low boiling point (such as at least one selected from thegroup consisting of a low-molecular alcohol with 1 to 4 carbon atoms, alow-molecular ketone with 3 to 5 carbon atoms, and an acetic acid alkylester having an alkyl group with 1 to 4 carbon atoms; in particular, atleast one selected from the group consisting of a low-molecular alcoholwith 1 to 4 carbon atoms, a low-molecular ketone with 3 to 5 carbonatoms, and an acetic acid alkyl ester having an alkyl group with 1 to 4carbon atoms, the low-molecular alcohol, low-molecular ketone, andacetic acid alkyl ester having a boiling point lower than that of water)is more preferable for allowing drying in a short time and facilitatingeven more uniform application on the culturing surface. Methanol andethanol are particularly preferable for their low cost and excellentoperability.

One type of these solvents may be used alone, or a combination of two ormore types may be used.

Since the solvent has excellent solubility with respect to atemperature-responsive polymer, the temperature-responsive polymer tendsnot to become insoluble and precipitate even at a temperature equal toor greater than the cloud point (such as room temperature or 37° C.).This eliminates the need to manage the temperature of thetemperature-responsive polymer during application of thetemperature-responsive polymer, allowing easy preparation of a coatedculturing surface.

In the coated culturing surface preparation step I, it may be preferableto include a hydrophilic molecule in the temperature-responsive polymersolution to facilitate self-aggregation of cells depending on the typeof cells to be used, such as when using highly adhesive mesenchymalcells or cancer cells with low aggregation strength. Examples of thehydrophilic molecule include non-ionic, hydrophilic molecules that donot affect the C/A ratio of the temperature-responsive polymer, such aspolyethylene glycol (PEG), dimethyl acrylamide (DMAA), glycerin, TritonX, polypropylene glycol, and the like.

In the coated culturing surface preparation step I, the content of thetemperature-responsive polymer in the temperature-responsive polymersolution is preferably 0.0010 mass % to 3.0 mass %, more preferably0.0012 mass % to 2.5 mass %, relative to the temperature-responsivepolymer solution (100 mass %) to facilitate uniform coating of theculturing surface by the temperature-responsive polymer.

In the coated culturing surface preparation step I, the content of thehydrophilic molecule in the temperature-responsive polymer solution ispreferably 0.00001 mass % to 0.00015 mass %, more preferably 0.00003mass % to 0.0001 mass %, relative to the temperature-responsive polymer(100 mass %) to facilitate self-aggregation of cells.

In the coated culturing surface preparation step I, thetemperature-responsive polymer solution may be applied to the entireculturing surface or to a portion of the culturing surface. When thetemperature-responsive polymer solution is applied to a portion of theculturing surface, one coated culturing surface or a plurality of coatedculturing surfaces may be provided on the culturing surface. A cellculture container with a cell non-adhesive culturing surface ispreferably used when the temperature-responsive polymer solution isapplied to a portion of the culturing surface.

In the coated culturing surface preparation step I, preferred conditionsfor drying the applied temperature-responsive polymer solution aredrying under atmospheric pressure at a temperature of 10° C. to 70° C.for 1 to 3,000 minutes to uniformly coat the culturing surface with thetemperature-responsive polymer or the temperature-responsive polymercomposition. Quick drying of the applied temperature-responsive polymersolution facilitates uniform coating on the culturing surface with aneven distribution of the temperature-responsive polymer or thetemperature-responsive polymer composition.

The applied temperature-responsive polymer solution may, for example, bedried by letting the cell culture container stand in an incubator at 37°C. or 40° C.

In the coated culturing surface preparation step II, examples of thesolvent for dissolving the temperature-responsive polymer or thetemperature-responsive polymer composition include water; physiologicalsaline; and buffer solutions such as a phosphate buffer solution,phosphate buffered saline (PBS), and a tris buffer solution.

In the coated culturing surface preparation step II, examples of themethod of cooling the temperature-responsive polymer aqueous solutioninclude placing the temperature-responsive polymer aqueous solution in arefrigerator at approximately 4° C. and cooling to a temperature at orbelow the cloud point.

In the coated culturing surface preparation step II, examples of themethod of casting the temperature-responsive polymer aqueous solutiononto the culturing surface include a method of tilting the culturingsurface to spread the temperature-responsive polymer aqueous solutionthat has a temperature at or below the cloud point and a method ofspreading the temperature-responsive polymer aqueous solution using aspatula.

In the coated culturing surface preparation step II, examples of themethod of heating the cast temperature-responsive polymer aqueoussolution to above the cloud point include a method of letting theculturing surface after the casting step stand in an incubator at 37° C.

[Production Method of Three-Dimensional Tissue Body]

The above-described production apparatus of a three-dimensional tissuebody of Embodiment (III) is used in a production method of athree-dimensional tissue body of Embodiment (III).

The production method of a three-dimensional tissue body of Embodiment(III) includes a seeding step of seeding at least one type of cells onthe coated culturing surface (also referred to as the “first seedingstep” in Aspect (III)) and a culturing step of culturing the seededcells to obtain a ringed three-dimensional tissue body wound around theshaft (also referred to as the “first culturing step” in Aspect (III)).

In Aspect (III), the three-dimensional tissue body, wound around theshaft, that is obtained in the first seeding and culturing step is alsoreferred to as a “ringed three-dimensional tissue body”, and thethree-dimensional tissue body in which a plurality of ringedthree-dimensional tissue bodies are stacked is also referred to as a“luminal three-dimensional tissue body”.

Operations of the first seeding step and the first culturing step may becontrolled by a computer or the like to be performed automatically,since operations performed automatically may be more aseptic andhygienic by virtue of not being performed by hand.

(First Seeding Step)

Examples of the cells seeded in the seeding step include vascular cellssuch as vascular endothelial cells and vascular smooth muscle cells,cardiomyocytes, chondrocytes, nerve cells, adipocytes, adipose stemcells, hepatocytes, fibroblasts, renal cells, smooth muscle cells, iPScells, and ES cells. When manufacturing a three-dimensional tissue bodyhaving extracellular matrix as the principal component, the cells arepreferably vascular cells such as vascular endothelial cells andvascular smooth muscle cells, cardiomyocytes, chondrocytes, nerve cells,adipocytes, adipose stem cells, hepatocytes, fibroblasts, renal cells,or smooth muscle cells and are more preferably fibroblasts ormesenchymal cells. One type of these cells may be used alone, or acombination of two or more types may be used.

In particular, use of vascular endothelial cells and smooth muscle cellsallows a synthetic blood vessel to be obtained. Use of chondrocytes andfibroblasts allows a synthetic trachea to be obtained. Established cells(such as COS cells) that have a promoter such as an SV40 promoter, havean introduced expression vector incorporating genes expressing proteinsconstituting extracellular matrix such as elastin or collagen, andexpress a large T antigen may be used when manufacturing athree-dimensional tissue body including material secreted by cells, suchas substances constituting extracellular matrix. Use of the establishedcells such as COS cells allows support of a copy of the introducedgenes, and a large amount of gene expression allows efficientmanufacturing of a three-dimensional tissue body having extracellularmatrix as the principal component.

In the seeding step, the density of all of the seeded cells on thecoated culturing surface is preferably a confluency of 90% to 100%relative to the surface area of the coated culturing surface, morepreferably a confluency of 95% to 100%, and even more preferably aconfluency of 99% to 100%. The properties of the seeded cells may changeduring growth. Setting the density of seeded cells within theaforementioned ranges makes it more difficult for the seeded cells togrow and allows formation of a ringed three-dimensional tissue bodywound around the shaft before cells grow, thereby allowing formation ofa three-dimensional tissue body that includes cells with the sameproperties as at the time of seeding.

While the density of all of the seeded cells on the coated culturingsurface depends on the type of cell, a density of 20 cells/mm² to 15,000cells/mm² is preferred. Live cells are seeded.

In the seeding step, a wall surface may be provided on the outer edge ofthe culturing surface to prevent seeded cells from being dispersed belowthe culturing surface and to facilitate adhesion of the cells to thecoated culturing surface. The production apparatus of athree-dimensional tissue body of Embodiment (III) may be placed inside acontainer with a slightly larger inner diameter than the outer diameterof the culturing surface (see FIG. 21). As a result, seeded cells tendnot to fall below the culturing surface, and seeded cells can be causedto adhere to the coated culturing surface. The difference between theouter diameter of the culturing surface and the inner diameter of thecontainer is preferably 15.0 mm or less to prevent seeded cells fromfalling through the gap and so as not to block expansion of the medium.

The production apparatus of a three-dimensional tissue body may be leftto stand in an incubator at 37° C. and subsequently removed and placedon a clean bench at room temperature, for example, with cell seedingthen being performed.

Cells are preferably seeded after being diluted in a medium. The mediumfor dilution may be any medium in which cells can be cultured.

(First Culturing Step)

The conditions for culturing the seeded cells may, for example, be theuse of a typical cell incubator at 37° C. The cells are preferablycultured continuously until obtaining a ringed three-dimensional tissuebody wound around the shaft. Specifically, the cells are preferablycultured for 10 hours to 96 hours and more preferably for 15 hours to 48hours.

The cells adhered to and cultured on the coated culturing surfaceself-aggregate towards the inside of the coated culturing surface andwind around the shaft in the form of a ring. The ringedthree-dimensional tissue body wound around the shaft has living cellsinside the three-dimensional tissue body.

Examples of the ringed three-dimensional tissue body wound around theshaft include a three-dimensional tissue body with cells as theprincipal component and may be a three-dimensional tissue body composedof cells. The ringed three-dimensional tissue body wound around theshaft may be a three-dimensional tissue body with extracellular matrixas the principal component, the extracellular matrix being constructedby secretion of proteins or the like that constitute extracellularmatrix. The ringed three-dimensional tissue body wound around the shaftmay be a three-dimensional tissue body including substances secretedfrom cells (for example, proteins such as those constitutingextracellular matrix), a three-dimensional tissue body having substancessecreted from cells as the principal component, or a three-dimensionaltissue body composed only of substances secreted from cells. Examples ofthe substances secreted from cells include proteins, sugars, and lipids,with proteins being preferred.

“Having as a principal component” refers to exceeding 50 mass % relativeto the mass of the three-dimensional tissue body (100 mass %), with 60mass % or more being preferable and 70 mass % or more being morepreferable.

The three-dimensional tissue body is preferably obtained by seedingcells on all of the coated culturing surfaces and culturing the seededcells.

(Culturing Surface Moving Step)

The production method of a three-dimensional tissue body of Embodiment(III) preferably further includes repeating a culturing surface movingstep of moving the culturing surface in the extending direction of theshaft after obtaining the ringed three-dimensional tissue body woundaround the shaft, a seeding step of seeding at least one type of cellson the coated culturing surface after the culturing surface is moved(also referred to as the “second and subsequent seeding steps” in Aspect(III)), and a culturing step of culturing the seeded cells to obtainanother ringed three-dimensional tissue body wound around the shaftadjacent to the ringed three-dimensional tissue body wound around theshaft (also referred to as the “second and subsequent culturing steps”in Aspect (III)). In other words, after the first seeding step and thefirst culturing step are performed to obtain a ringed three-dimensionaltissue body, the production method may include repetition of theculturing surface moving step, the second and subsequent seeding steps,and the second and subsequent culturing steps.

Operations of the culturing surface moving step, the second andsubsequent seeding steps, and the second and subsequent culturing stepsmay be controlled by a computer or the like to be performedautomatically, since operations performed automatically may be moreaseptic and hygienic by virtue of not being performed by hand.

The culturing surface moving step is a step provided after the ringedthree-dimensional tissue body wound around the shaft is obtained in theprevious seeding step. The culturing surface moving step may be providedimmediately after the ringed three-dimensional tissue body wound aroundthe shaft is obtained in the previous culturing step or after aninterval (such as 1 minute to 96 hours).

In the culture face moving step, the distance which the culturingsurface is moved in the extending direction of the shaft is preferably0.01 mm to 50 mm and more preferably 0.1 mm to 10 mm.

The cells of the ringed three-dimensional tissue body obtained throughthe first seeding step and the first culturing step and the cells of theringed three-dimensional tissue body obtained through the second seedingstep and the second culturing step may be adhered to each other orseparated. The distance between two ringed three-dimensional tissuebodies that are separated may, for example, be the aforementioneddistance by which the culturing surface is moved in the extendingdirection of the shaft. Two ringed three-dimensional tissue bodies thatare separated can easily be connected as follows: by the cells in theringed three-dimensional tissue bodies expanding and contracting,migrating, or the like so that the cells in two ringed three-dimensionaltissue bodies adhere to each other and connect the two ringedthree-dimensional tissue bodies; by two ringed three-dimensional tissuebodies connecting via a substance (such as proteins) secreted by thecells in the two ringed three-dimensional tissue bodies; by addition ofseparately produced proteins, cells, or the like between two ringedthree-dimensional tissue bodies for the two ringed three-dimensionaltissue bodies to connect; by connecting two ringed three-dimensionaltissue bodies with a combination of these methods; and the like. Theculturing surface may be moved by fixing the culturing surface andmoving the shaft, by fixing the shaft and moving the culturing surface,or by moving both the culturing surface and the shaft.

One method of controlling the thickness of the three-dimensional tissuebody is, for example, to provide the second and subsequent seeding stepsand the second and subsequent culturing steps, or to provide repetitionof the second and subsequent seeding steps and the second and subsequentculturing steps, without providing the culturing surface moving step,thereby winding another ringed three-dimensional tissue body around theringed three-dimensional tissue body, wound around the shaft, that wasobtained in the previous seeding step to make a portion of thethree-dimensional tissue body thicker.

After winding another ringed three-dimensional tissue body around theringed three-dimensional tissue body wound around the shaft, theculturing surface moving step may be provided and similar operations maybe continued to increase the thickness of the entire three-dimensionaltissue body. A three-dimensional tissue body with layers of differentcells can be obtained by using different types of cells to form theringed three-dimensional tissue body wound around the shaft and theringed three-dimensional tissue body wound therearound.

(Second and Subsequent Seeding Steps)

Examples of the second and subsequent seeding steps include a stepsimilar to the first seeding step.

The type, concentration, and the like of cells used in the second andsubsequent seeding steps may be the same as or different from those ofthe first seeding step. The type, concentration, and the like of cellsused in the second and subsequent seeding steps may be the same ordifferent in each of the second and subsequent seeding steps.

(Second and Subsequent Culturing Steps)

Examples of conditions for culturing seeded cells include conditionssimilar to those of the above-described first culturing step.

The conditions and the like of the second and subsequent culturing stepsmay be the same as or different from those of the first culturing step.The conditions and the like of the second and subsequent culturing stepsmay be the same or different in each of the second and subsequentculturing steps.

By performance of the second and subsequent culturing steps, a newringed three-dimensional tissue body wound around the shaft (new ringedthree-dimensional tissue body) is formed adjacent to the ringedthree-dimensional tissue body, wound around the shaft, obtained in theprevious culturing step (previous ringed three-dimensional tissue body).The previous and new ringed three-dimensional tissue bodies may, forexample, be adhered to each other by culturing at 37° C. for one hour to30 days to obtain a luminal three-dimensional tissue body.

The number of repetitions of the culturing surface moving step, thesecond and subsequent seeding steps, and the second and subsequentculturing steps may be appropriately selected in accordance with thethickness and length of the laminal three-dimensional tissue body. Forexample, 1 to 20 repetitions are preferable, and 1 to 10 repetitions aremore preferable.

When manufacturing a three-dimensional tissue body that includessubstances (in particular proteins) secreted by cells seeded in theseeding step, such as a three-dimensional tissue body includingextracellular matrix, a step of removing cells included in thethree-dimensional tissue body may be provided after the culturing step.Methods of removing cells included in a three-dimensional tissue bodyinclude, for example, killing cells by treating with high-pressuretreatment, alcohol treatment, surfactant treatment, or the like orculturing under conditions in which cells do not easily survive.Provision of the step of removing cells included in thethree-dimensional tissue body allows at least a portion of the cells inthe three-dimensional tissue body to be removed.

Furthermore, a plurality of types of cells may be used to manufacture athree-dimensional tissue body, and particular cells alone may be removed(for example, COS cells alone may be removed from a three-dimensionaltissue body that includes chondrocytes and COS cells incorporating genesexpressing proteins constituting extracellular matrix). Examples ofmethods of removing only particular cells include culturing underconditions in which only particular cells can survive or do not easilysurvive, such as increasing the sensitivity of cells to antibiotics andculturing in a medium including antibiotics, or providing only the cellsto be maintained in the three-dimensional tissue body with resistance toantibiotics and culturing in a medium including antibiotics.

The three-dimensional tissue body obtained with the production method ofa three-dimensional tissue body of Embodiment (III) may be a ringedthree-dimensional tissue body or a luminal three-dimensional tissuebody. The inner diameter of the three-dimensional tissue body ispreferably 0.01 mm to 100 mm and more preferably 0.1 mm to 50 mm. Theouter diameter of the three-dimensional tissue body is preferably 0.1 mmto 120 mm and more preferably 0.2 mm to 70 mm. The length of the luminalthree-dimensional tissue body is preferably 0.1 mm to 300 mm and morepreferably 1 mm to 250 mm.

The three-dimensional tissue body obtained by the production method of athree-dimensional tissue body of Embodiment (III) may be athree-dimensional tissue body with cells as the principal component or athree-dimensional tissue body including material secreted by cells, suchas a three-dimensional tissue body with extracellular matrix as theprincipal component.

Examples of preferred conditions for producing a three-dimensionaltissue body with extracellular matrix as the principal component includei) using cells highly capable of producing cell matrix such as collagen,laminin, fibronectin, elastin, or the like (for example, fibroblasts ormesenchymal cells), ii) adding ascorbic acid that promotes theproduction of extracellular matrix to the medium, iii) reducing theseeding density of cells to increase the ratio of extracellular matrixto cells, iv) increasing the production (secretion) amount ofextracellular matrix by increasing the time for culturing thethree-dimensional tissue body with a ringed shape, a luminal shape, orthe like wound around the shaft (for example, culturing for 24 to 350hours, preferably 48 to 170 hours, in the first culturing step and thesecond culturing step) and maturing the bonds of the extracellularmatrix (for example, the bonds of collagen fibers), v) using a shaftwith holes on the surface to stably provide nutrients and oxygen tocells to facilitate the diffusion of cell metabolites in the medium,thereby promoting production of extracellular matrix, and vi) increasingthe movement distance of the culturing surface in the culturing surfacemoving step to reduce the cell density in the extending direction of theshaft.

Examples of the resulting three-dimensional tissue body withextracellular matrix as the principal component include collagenousluminal biotubes usable in synthetic blood vessels, synthetic tracheae,and the like.

Examples of the three-dimensional tissue body of Embodiment (III)include three-dimensional tissue bodies usable in a synthetic bloodvessel, a synthetic trachea, and the like, such as a three-dimensionaltissue body including cells seeded in the seeding step; athree-dimensional tissue body including substances secreted by cells,such as a three-dimensional tissue body having proteins secreted bycells as the principal component, a three-dimensional tissue bodyincluding extracellular matrix, and a three-dimensional tissue bodyhaving extracellular matrix as the principal component; and the like.

Examples of a three-dimensional tissue body usable in a synthetic bloodvessel include a three-dimensional tissue body obtained with thefollowing method.

After obtaining a ringed three-dimensional tissue body of vascularendothelial cells around a shaft in the first seeding step and firstculturing step (such as a thick ringed three-dimensional tissue body ofvascular endothelial cells obtained by continuously performing the stepsof seeding and culturing vascular endothelial cells 2 to 3 times), aringed three-dimensional tissue body of smooth muscle cells (such as athick three-dimensional tissue body of smooth muscle cells obtained bycontinuously performing the steps of seeding and culturing smooth musclecells 3 to 30 times) may be wound around the ringed three-dimensionaltissue body of vascular endothelial cells in the second seeding step andsecond culturing step to yield a double-layered ringed three-dimensionaltissue body having a layer of vascular endothelial cells around theshaft and a layer of smooth muscle cells on the outside of the layer ofvascular endothelial cells. Furthermore, a culturing surface moving stepmay be provided and similar operations performed to yield adouble-layered ringed or luminal three-dimensional tissue body having astructure similar to that of a blood vessel in a living organism, suchas a blood vessel having a layer of vascular endothelial cells on theinner wall and a layer of smooth muscle cells along the periphery of thelayer of vascular endothelial cells. To prevent the cells forming eachlayer from mixing and to adjust the strength and flexibility of theblood vessel, a layer that has collagen, elastin, or the like as theprincipal component (in particular, a layer having elastin as theprincipal component) and that functions as a barrier preventing thevascular endothelial cells and smooth muscle cells from mixing can alsobe provided between the layer of vascular endothelial cells and thelayer of smooth muscle cells in the three-dimensional tissue body by,for example, a method such as providing a step of seeding and culturingcells that secrete collagen or elastin, e.g. mesenchymal stem cells,fibroblasts, or the like, between the step of forming the layer ofvascular endothelial cells and the step of forming the layer of smoothmuscle cells; or covering the layer of vascular endothelial cells with acollagen or elastin tube (such as a three-dimensional tissue body,manufactured with the manufacturing method of a three-dimensional tissuebody of Embodiment (III), including proteins secreted by cells).Provision of the aforementioned layer functioning as a barrier, such asthe inner elastic plate found in blood vessels of living organisms,prevents migratory cells from moving between layers after forming thethree-dimensional tissue body, yielding a three-dimensional tissue bodywith a structure even closer to that of a blood vessel in a livingorganism.

Examples of a three-dimensional tissue body usable in a synthetictrachea include a three-dimensional tissue body obtained with thefollowing method.

A culturing surface moving step is provided after obtaining a ringedthree-dimensional tissue body (a) of chondrocytes by the first seedingstep and first culturing step, and a new ringed three-dimensional tissuebody (b) of fibroblasts is formed adjacent to the ringedthree-dimensional tissue body of chondrocytes by the second seeding stepand second culturing step. By appropriately repeating the above process,a luminal three-dimensional tissue body in which ringedthree-dimensional tissue bodies are stacked in the order a-b-b-a-b-b orthe like in the extending direction of the shaft, for example, can beobtained. A ringed three-dimensional tissue body having extracellularmatrix as the principal component, a three-dimensional tissue bodyincluding substances secreted by cells, a ringed three-dimensionaltissue body including cells that excrete substances such as thecomponents constituting extracellular matrix, or the like may beprovided between the ringed three-dimensional tissue bodies. To adjuststrength, the resulting luminal three-dimensional tissue body may becovered with a collagen tube or an elastin tube (for example, athree-dimensional tissue body, manufactured with the manufacturingmethod of a three-dimensional tissue body of Embodiment (III), includingproteins secreted by cells).

Even when using only ringed three-dimensional tissue bodies ofchondrocytes, the ringed three-dimensional tissue bodies of chondrocytescan be connected by extracellular matrix secreted from the chondrocytesto form a synthetic trachea. To strengthen the synthetic trachea andencourage blood vessels to work themselves in after grafting, thesynthetic trachea preferably includes a ringed three-dimensional tissuebody of chondrocytes and a ringed three-dimensional tissue body offibroblasts.

An example of a three-dimensional tissue body including substancessecreted by cells is a three-dimensional tissue body, obtained by thefollowing method, that includes substances such as proteins (forexample, proteins constituting extracellular matrix).

COS cells that have an SV40 promoter sequence and have an introducedexpression vector incorporating genes expressing proteins constitutingextracellular matrix such as elastin or collagen are used to form aringed or luminal three-dimensional tissue body, and the COS cells arecaused to secrete proteins or the like. After the culturing step, theCOS cells are killed with a method such as killing the cells by treatingwith high-pressure treatment, alcohol treatment, surfactant treatment,or the like or culturing under conditions in which COS cells do noteasily survive (for example, increasing the sensitivity of the COS cellsto antibiotics and culturing the COS cells in a medium includingantibiotics). The COS cells are then removed. The three-dimensionaltissue body of Embodiment (III) may be a three-dimensional tissue bodyincluding dead cells that have not been completely removed, survivingcells, or the like.

The resulting three-dimensional tissue body including proteinsconstituting extracellular matrix may, for example, be used as a coatingmaterial to strengthen the three-dimensional tissue body obtained withthe manufacturing method of Embodiment (III), a synthetic blood vessel,a myocardial patch, prosthetic material, or the like.

An example of a production method of a three-dimensional tissue body ofEmbodiment (III) is described below with reference to FIG. 22, FIG. 23,and FIG. 27.

FIG. 22 is an outline of an example production method of athree-dimensional tissue body of Embodiment (III).

With a shaft inserted through a through hole of a culturing surface in aproduction apparatus of a three-dimensional tissue body (see (i) of FIG.22), a temperature-responsive polymer is applied to the culturingsurface to coat the culturing surface, thereby preparing a coatedculturing surface (see (ii) of FIG. 22). In this example, the culturingsurface and the shaft are in contact. Subsequently, the productionapparatus of the three-dimensional tissue body is placed inside acontainer having an inner diameter slightly larger than the outerdiameter of the culturing surface and is immersed in a medium. Cells arethen seeded (first seeding step; see (iii) of FIG. 22). The seeded cellsadhere to the coated culturing surface (see (iv) of FIG. 22). In thisexample, the density of cells is 100% confluency. Subsequently, thecells adhered to the coated culturing surface start to aggregate towardsthe central portion of the coated culturing surface, and the edgeseparates from the coated culturing surface and starts to warp, yieldinga cell structure with a warped edge (see (v) of FIG. 22). The cells thenfurther aggregate to form a ringed shape, yielding a ringedthree-dimensional tissue body wound around the shaft (see (vi) of FIG.22). The ringed three-dimensional tissue body wound around the shaft iswound around the shaft to a degree preventing the tissue body fromfalling off the shaft. Therefore, even when the shaft is not celladhesive, the position at which the three-dimensional tissue body iswound during production does not change greatly. Subsequently, theculturing surface is moved downward in the extending direction of theshaft to provide a gap, adjacent to the bottom of the resulting ringedthree-dimensional tissue body, for forming another ringedthree-dimensional tissue body (culturing surface moving step; see (vii)of FIG. 22). Cells are then seeded again (second seeding step; see(viii) of FIG. 22) and cultured to obtain a luminal three-dimensionaltissue body in which ringed three-dimensional tissue bodies are stacked.A long, luminal three-dimensional tissue body can be obtained byrepeating the culturing surface moving step, the second and subsequentseeding steps, and the second and subsequent culturing steps (see (ix)of FIG. 22).

In the above example, a three-dimensional tissue body havingextracellular matrix as the principal component can be obtained by, forexample, reducing the number of seeded cells, leaving the ringedthree-dimensional tissue body wound around the shaft to stand andwaiting for secretion of proteins constituting extracellular matrix, orthe like. Some or all of the above steps may be controlled by a computeror the like to be performed automatically.

FIG. 23 is an outline of an example production method of athree-dimensional tissue body of Embodiment (III) using a productionapparatus of a three-dimensional tissue body having a gap between theculturing surface and the shaft.

With a shaft inserted through a through hole of a culturing surface in aproduction apparatus of a three-dimensional tissue body (see (i) of FIG.23), a temperature-responsive polymer is applied to the culturingsurface to coat the culturing surface, thereby preparing a coatedculturing surface (see (ii) of FIG. 23). In this example, the diameterof the shaft is smaller than the hole diameter of the through hole, thusforming a gap between the culturing surface and the shaft. Subsequently,the production apparatus of the three-dimensional tissue body is placedinside a container having an inner diameter slightly larger than theouter diameter of the culturing surface and is immersed in a medium.Cells are then seeded (first seeding step; see (iii) of FIG. 23). Theseeded cells adhere to the coated culturing surface (see (iv) of FIG.23). In this example, the density of cells is 100% confluency.Subsequently, the cells adhered to the coated culturing surface start toaggregate towards the central portion of the coated culturing surface,and the edge separates from the coated culturing surface and starts towarp, yielding a cell structure with a warped edge (see (v) of FIG. 23).The cells then further aggregate to form a ringed shape, and theaggregating cell structure jumps over the gap between the culturingsurface and the shaft to wind around the shaft and form a ringedthree-dimensional tissue body (see (vi) of FIG. 23). Subsequently, theculturing surface is moved upward in the extending direction of theshaft to provide a gap, adjacent to the top of the resulting ringedthree-dimensional tissue body, for forming another ringedthree-dimensional tissue body (culturing surface moving step; see (vii)of FIG. 23). Cells are then seeded again (second seeding step; see(viii) of FIG. 23) and cultured to obtain a luminal three-dimensionaltissue body in which ringed three-dimensional tissue bodies are stacked.A long, luminal three-dimensional tissue body can be obtained byrepeating the culturing surface moving step, the second and subsequentseeding steps, and the second and subsequent culturing steps (see (ix)of FIG. 23).

Some or all of the above steps may be controlled by a computer or thelike to be performed automatically.

FIG. 27 is an outline of an example production method of athree-dimensional tissue body of Embodiment (III) using a productionapparatus of a three-dimensional tissue body having a plurality ofculturing surfaces and a gap between the culturing surfaces and theshaft.

With one shaft inserted through a through hole of each of two culturingsurfaces in a production apparatus of a three-dimensional tissue body(see (i) of FIG. 27), a temperature-responsive polymer is applied to thetwo culturing surfaces to coat the two culturing surfaces, therebypreparing coated culturing surfaces (see (ii) of FIG. 27). In thisexample, the diameter of the shaft is smaller than the hole diameter ofthe through hole, thus forming a gap between each culturing surface andthe shaft. Subsequently, the production apparatus of a three-dimensionaltissue body is placed inside a container having an inner diameter largerthan the outer diameter of the culturing surfaces and is immersed in amedium. Cells are then seeded (first seeding step; see (iii) of FIG.27). The seeded cells adhere to the coated culturing surfaces (see (iv)of FIG. 27). In this example, the density of cells is 100% confluency.Subsequently, the cells adhered to the coated culturing surface start toaggregate towards the central portion of the coated culturing surface,and the edge separates from the coated culturing surface and starts towarp, yielding a cell structure with a warped edge (see (v) of FIG. 27).The cells then further aggregate to form a ringed shape, and theaggregating cell structure jumps over the gap between the culturingsurface and the shaft to wind around the shaft and form a ringedthree-dimensional tissue body (see (vi) of FIG. 27). Subsequently, thetwo ringed three-dimensional tissue bodies are cultured and connect toeach other to yield a luminal three-dimensional tissue body (see (vii)of FIG. 27).

In (vii) of FIG. 27, the culturing surface is omitted to illustrate theluminal three-dimensional tissue body more clearly. Since a gap existsbetween the culturing surfaces and the shaft in FIG. 27, the two ringedthree-dimensional tissue bodies connect to yield a luminalthree-dimensional tissue body whether or not the culturing surfaces arepresent.

Furthermore, a longer luminal three-dimensional tissue body can beobtained by methods such as increasing the number of culturing surfacesor providing a culturing surface moving step, second and subsequentseeding steps, and second and subsequent culturing steps.

Some or all of the above steps may be controlled by a computer or thelike to be performed automatically.

[[Aspect (IV)]]

(Manufacturing Method of a Cell Structure)

A manufacturing method of a cell structure in an embodiment of Aspect(IV) (manufacturing method of Embodiment (IV)) is a method of producinga coated region in which a culturing surface is coated with atemperature-responsive polymer or a temperature-responsive polymercomposition, forming a droplet of a cell suspension in the coatedregion, and performing cell culturing in the droplet.

As illustrated in greater detail in FIG. 32, an example manufacturingmethod of Embodiment (IV) includes preparing a culturing surface (see(i) of FIG. 32), then disposing a temperature-responsive polymer or atemperature-responsive polymer composition on the culturing surface (see(ii) of FIG. 32), coating the culturing surface with thetemperature-responsive polymer or the temperature-responsive polymercomposition to produce a coated region (see (iii) of FIG. 32), forming adroplet of cell suspension in the coated region (see (iv) of FIG. 32),performing cell culturing in the droplet (see (v) of FIG. 32), andforming a cell structure (see (vi) of FIG. 32).

Here, in the manufacturing method of Embodiment (IV), it is essentialthat the surface zeta potential of the coated region produced on theculturing surface be 0 mV to 50 mV to obtain the effect of spontaneousformation of a cell structure, as described below.

The effects of the manufacturing method of Embodiment (IV) are nowdescribed.

A hanging drop method known as a manufacturing method of a cellstructure manufactures a spheroidal cell structure in a droplet byproducing a droplet of a cell suspension at the tip of a tubular memberand holding the droplet for a predetermined time period (such asapproximately 2 weeks) while maintaining the spherical shape of thedroplet using the surface tension of the droplet. The operation to holdthe droplet is complicated, however, making it difficult to manufacturea spheroidal cell structure easily.

By contrast, the manufacturing method of Embodiment (IV) allows adroplet to be held in the state when placed on a culturing surface,thereby allowing a spheroidal cell structure to be manufactured easily.

Another possible method would be to use a cell culture containerprovided with one or a plurality of wells partitioned by a culturingsurface and a wall surface to produce a coated region and a non-coatedregion, separated from the coated region, on the culturing surface ofthe well. A cell suspension is then added to the well. The manufacturingmethod of Embodiment (IV) has the following effects as compared to sucha method.

In the above-described method, the cell suspension is added to the cellculture container so as to immerse both the coated region and thenon-coated region in the cell suspension. Seeded cells may thereforeadhere to the non-coated region. At this time, the cells in thenon-coated region might obstruct formation of a spheroidal cellstructure with a well-defined shape in the coated region. Therefore,when using a cell culture container with a wall surface, it may becomenecessary to treat all or a portion of the culturing surface to becomecell non-adhesive. Such treatment may also cause elution of cytotoxicsubstances into the medium.

By contrast, the droplet of cell suspension in the coated region isisolated from the non-coated region in the manufacturing method ofEmbodiment (IV), making the aforementioned treatment to provide cellnon-adhesiveness unnecessary and reducing the likelihood of adverselyaffecting cell growth.

In the above-described method, cells adhere to the coated region afteraddition of the cell suspension to the well, whereas cells do not adhereto the non-coated region treated to be cell non-adhesive. The cells thatdo not adhere to the non-coated region might adversely affect the growthof cells adhered to the coated region by provoking apoptosis orproducing heat shock proteins. Therefore, with this method it may benecessary to remove the cells in the non-coated region from the well byan operation such as medium exchange.

By contrast, the need to remove cells in the non-coated region with themanufacturing method of Embodiment (IV) is small, because a droplet ofcell suspension is formed in the coated region, which is provided withcell adhesiveness by being coated with a temperature-responsive polymeror a temperature-responsive polymer composition, in isolation from thenon-coated region. Hence, the amount of cells used can be reduced.Furthermore, the need for operations such as medium exchange is small,allowing a reduction in the amount of medium used. Overall, themanufacturing method of Embodiment (IV) allows a reduction inmanufacturing costs.

In the manufacturing method of Embodiment (IV), an automatic cellculturing apparatus used in a 384 well cell culture plate or the likemay be used (for example, a droplet of cell suspension can be ejectedonto a culturing surface, which is coated with a temperature-responsivepolymer or a temperature-responsive polymer composition, by a programfor injecting a cell suspension onto multiple plates using 16 nozzles).Devices normally used in the present technical field, such as analysisdevices for cell culturing plates, may also be used. Therefore, themanufacturing method of Embodiment (IV) can reduce the manufacturingcosts of cell structures while also achieving an economic effect bybroadening the uses of the aforementioned apparatuses and devices.

As described above, it is essential that the surface zeta potential ofthe coated region be 0 mV to 50 mV in the manufacturing method of a cellstructure in an embodiment of Aspect (IV). Setting the surface zetapotential to 0 mV or greater allows adhesion of negatively chargedcells, while setting the surface zeta potential to 50 mV or less canreduce cytotoxicity.

For similar reasons, the surface zeta potential is preferably from 0 mVto 35 mV and more preferably from 10 mV to 25 mV.

The surface zeta potential can be adjusted by adjusting the C/A ratio inthe temperature-responsive polymer or the temperature-responsive polymercomposition.

A culturing surface that has a surface zeta potential in theaforementioned particular range allows a cell structure (spheroid)having an aggregated (pellet-like) structure to be formed easily bysimply culturing cells under appropriate culture conditions.

The reason is that setting the surface zeta potential within theaforementioned particular range is inferred to provide the culturingsurface with a weak positive charge that does not trigger cytotoxicity,to ensure rapid adhesion of the seeded cells, to improve cell activityand encourage secretion of extracellular matrix, and also toappropriately inhibit cell migration, strengthening the bond betweencells.

The phenomenon of forming a cell structure on a culturing surface havinga surface zeta potential in the aforementioned particular range isextremely reproducible, allowing production of homogenous cellstructures.

The mass of cells produced using a known cell culture container issimply a collection of cells that cannot adhere to a culturing surfacethat is cell non-adhesive, leading to the problem of low viability ofthe cells constituting the cell mass.

A cell structure (spheroid) manufactured with the manufacturing methodof Embodiment (IV), however, is formed after the process of cellsrapidly adhering to the culturing surface and expanding while growing. Arich extracellular matrix is thus produced between these cells,providing the cell structure itself with an extremely high viability(activity).

With the manufacturing method of Embodiment (IV), the experimenter canappropriately determine the size of the cell structures in accordancewith purpose and can either provide the size with a distribution or makethe size uniform.

The cell structure can be minute in size (for example, several hundredμm or less) or may have a diameter of 50 μm to 1,500 μm, with a diameterof 50 μm to 200 μm being preferable.

Since the manufacturing method of Embodiment (IV) allows production ofcell structures on a flat plate culturing surface, the culture containercan be observed directly with a microscope when performing an assay onthe cell structures produced using the cell culture container.

The manufacturing method of Embodiment (IV) also makes it possible forcells not to adhere to the outer edge of the culturing surface, which isthe border between the culturing surface and the wall of the cellculture container, allowing the size to be kept homogeneous for anextended period of time.

Each step in the manufacturing method of a cell culture container in anembodiment of Aspect (IV) is described below in detail.

In an example manufacturing method of Embodiment (IV), a culturingsurface is first prepared (see (i) of FIG. 32).

The culturing surface may be the culturing surface of a cell culturecontainer or the culturing surface of a material other than a cellculture container. Examples include commercially available plates,dishes, flasks, glass plates, silicone sheets, and the like.

The culturing surface may be cell adhesive or cell non-adhesive.

“Cell non-adhesive” refers to adherent cells (for example, fibroblasts,hepatocytes, vascular endothelial cells, and the like) either notadhering or tending not to adhere under normal culture conditions.

Examples of the material of the culturing surface include polystyrene,polyethylene terephthalate (PET), polypropylene, polybutene,polyethylene, and polycarbonate. Polystyrene and PET are preferable forbeing easy to mold precisely, for allowing adoption of varioussterilization methods, and for being suitable for microscope observationby virtue of being transparent.

A typical cell culture plate is made of plastic, such as polystyrene,that is surface treated (for example, plasma treated) for cells toadhere more easily to the culturing surface.

In the manufacturing method of a cell structure of Aspect (IV), itsuffices to prepare a culturing surface, and a cell culture containerneed not be used.

Next, a temperature-responsive polymer or a temperature-responsivepolymer composition is disposed on the culturing surface (see (ii) ofFIG. 32).

The temperature-responsive polymer and temperature-responsive polymercomposition disposed on the culturing surface in the manufacturingmethod of an embodiment of Aspect (IV) are now described in detail.

Examples of the temperature-responsive polymer andtemperature-responsive polymer composition in Aspect (IV) includetemperature-responsive polymers and temperature-responsive polymercompositions similar to those of Aspect (I), and similar polymers andpolymer compositions are preferred.

In Embodiment (IV), a manufacturing method of the temperature-responsivepolymer of (A-1) includes a production step of producing a mixturecontaining 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and anirradiation step of irradiating the mixture with ultraviolet light,where in the production step, the mixture further contains apolymerization inhibitor and water, and in the irradiation step, themixture is irradiated with ultraviolet light under an inert atmosphere.However, the manufacturing method is not limited to this example and maybe a different method.

In Embodiment (IV), the temperature-responsive polymer of (A-1) allowsformation of cell structures that have a luminal (tube-like) and anaggregated (pellet-like) structure by culturing cells under appropriateculture conditions, as described below.

In Embodiment (IV), a manufacturing method of the temperature-responsivepolymer of (A-2) includes a first polymerization step of irradiating afirst mixture containing 2-N,N-dimethylaminoethyl methacrylate (DMAEMA)with ultraviolet light, an adding step of adding an anionic monomer tothe first mixture at the point when the number-average molecular weightof the polymer in the first polymerization step reaches at least apredetermined value to produce a second mixture, and a secondpolymerization step of irradiating the second mixture with ultravioletlight. However, the manufacturing method is not limited to this exampleand may be a different method.

In Embodiment (IV), the temperature-responsive polymer composition of(C) may include a polymer of 2-N,N-dimethylaminoethyl methacrylate(DMAEMA) and/or a derivative thereof;2-amino-2-hydroxymethyl-1,3-propanediol; and one or more anionicsubstances selected from the group consisting of nucleic acids, heparin,hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylicacid, polymethacrylic acid, polyphosphoric acid, sulfatedpolysaccharide, curdlan, polyarginic acid, and alkali metal saltsthereof, as described above.

In Embodiment (IV), the DNA in the anionic substances listed in (C3) ispreferably DNA capable of inducing differentiation into differentiatedcells of stem cells such as iPS cells, ES cells, and mesenchymal stemcells, in particular cardiomyocytes, hepatocytes, nerve cells, andvascular endothelial cells.

In an example manufacturing method of Embodiment (IV), the coated regionmay be produced on the entire culturing surface (see (i), (ii) of FIG.33), or a plurality of coated regions may be produced on the culturingsurface (see (iii) of FIG. 33).

When producing a coated region on the entire culturing surface, atemperature-responsive polymer or a temperature-responsive polymercomposition solution may be cast on the culturing surface.

When producing a plurality of coated regions on the culturing surface,the temperature-responsive polymer or the temperature-responsive polymercomposition may be spotted by a technique using an eight-channelmicropipettor, by spotter printing to discharge solution quantitatively,by rotating screen drum printing, or by another such technique.

In particular, when producing a plurality of coated regions on theculturing surface, the area of each coated region is preferably 0.1 mm²to 30 mm². Adopting this range makes it easier to obtain the desiredminute spheroids.

The distance between coated regions is preferably from 0.1 mm to 10 mm.The “distance between coated regions” refers to the shortest distance onthe culturing surface between coated regions. Setting this distance to0.1 mm or greater can inhibit adhesion between the cells seeded inadjacent coated regions. Setting this distance to 10 mm or less allowsefficient production of a large quantity of cell structures (spheroidsor the like).

In particular, when producing a plurality of coated regions on theculturing surface, the number of coated regions may be set appropriatelyin accordance with the experimenter's purpose. The number may be 2 ormore, 10 or more, or 1,000 or more.

In particular, when producing a plurality of coated regions on theculturing surface, the planar shape of each coated region is preferablya circle, an ellipse, or another shape having the center of curvature onthe inside of the outer contour line.

The radius of curvature is preferably 0.1 mm to 50 mm and morepreferably 1 mm to 10 mm.

The diameter of a circular coated region is preferably 10 μm to 10 mmand more preferably 30 μm to 1,500 μm.

One of these shapes may be used alone, or a combination of two or moreshapes may be used.

In an example manufacturing method of Embodiment (IV), the amount oftemperature-responsive polymer per unit area in the coated regions ofthe culturing surface is preferably 5.0 ng/mm² to 50 ng/mm² and morepreferably 15 ng/mm² to 40 ng/mm². Adopting these ranges facilitatesachievement of the effect of easier formation of cell structures.

In an example manufacturing method of Embodiment (IV), the culturingsurface of the cell culture container may be coated with thetemperature-responsive polymer by techniques such as precipitation fromthe state of an aqueous solution, application of a solution and dryingof the solvent, exposure to radiation, exposure to low temperatureplasma, corona discharge, glow discharge, ultraviolet light, or graftpolymerization using a radical generator.

A method of coating the culturing surface with a temperature-responsivepolymer or a temperature-responsive polymer composition and a method ofspotting a temperature-responsive polymer or a temperature-responsivepolymer composition at a plurality of positions on the culturing surfacethat become the coated regions are described below.

The culturing surface in the manufacturing method of Embodiment (IV) maybe produced on the basis of a cell culture plate (for example, a 35 mmdish or the like) and can, for example, be used suitably in the field ofregenerative medicine.

The culturing surface may also be produced on the basis of a microplate(for example, a 96 hole plate or the like) and can, for example, be usedsuitably in the field of drug discovery (particularly drug screening).

Moisture may be removed from the temperature-responsive polymer or thetemperature-responsive polymer composition by heating or freeze drying,vacuum distillation, or the like, and the result may be dissolved in anorganic solvent, examples of which include methanol, ethanol, and otheralcohols; ketones; and esters. Among these solvents, methanol ispreferable for dissolution by virtue of having a low surface tension andboiling point, allowing rapid drying, and allowing more uniform coveringwith the temperature-responsive polymer. In the case of dissolution inan organic solvent, hydrophilic molecules that are non-ionic andhydrophilic, such as polyethylene glycol (PEG), dimethyl acrylamide(DMAA), glycerin, Triton X, polypropylene glycol, and the like may befurther added.

In an example of this manufacturing method, a coated region in which theculturing surface is coated with a temperature-responsive polymer or atemperature-responsive polymer composition is thus produced (see (iii)of FIG. 32).

In the manufacturing method of Embodiment (IV), it is essential that thesurface zeta potential of the coated region be 0 mV to 50 mV, preferably0 mV to 35 mV, and more preferably 10 mV to 25 mV, as described above.

In the manufacturing method of Embodiment (IV), the contact angle ofwater relative to the coated region of the culturing surface ispreferably from 50° to 90°, more preferably 60° to 80°, and particularlypreferably 620 to 780 to improve the effects of Aspect (IV).

The contact angle of water relative to the coated region refers to theaverage contact angle measured in conformity with JIS R3257 at severalarbitrary points within the coated region.

As described above, the coated region is preferably a region with apredetermined degree or higher of hydrophobicity to increase theinteraction with the cell surface and to increase the adhesiveness ofcells relative to the coated region.

The contact angle of water relative to the coated region of theculturing surface can be adjusted by adjusting the structure or amountof hydrophobic groups in the temperature-responsive polymer or thetemperature-responsive polymer composition.

In an example manufacturing method of Embodiment (IV), the bottom areaof the droplet in each coated region is preferably smaller than the areaof the coated region.

In the step of coating the culturing surface, the amount of thetemperature-responsive polymer or the temperature-responsive polymercomposition per unit area may become larger at the edge of the coatedregion than in the central portion of the coated region, and the edge ofthe coated region sometimes swells relative to the central portion. Inthis case, the shape of the cell structure formed by cells adhered tothe coated region tends to be difficult to control. Setting the bottomarea of the droplet to be smaller than the area of the coated regionmakes it possible to prevent cells from adhering to a portion of thecoated region in which the amount of the temperature-responsive polymeror the temperature-responsive polymer composition might not be uniform,thereby facilitating production of a cell structure with a well-definedshape.

In each coated region, the upper limit on the ratio of the bottom areaof the droplet to the area of the coated region may be 99% or less forthe same reasons as above, and the upper limit is preferably 97%, 95%90%, 80%, 70%, 60%, or 50%.

The lower limit on this ratio may be 10% or more, and the lower limit ispreferably 20% or 30%. Setting this ratio to 10% or more allowsefficient production of a sufficiently large cell structure.

In an example of a cell culture method using a cell culture container ofan embodiment of Aspect (IV), a droplet of cell suspension is formed inthe coated region (see (iv) of FIG. 32).

The manufacturing method of Embodiment (IV) can be suitably adopted inparticular for cells for which the diameter of the manufactured cellstructure is required to be highly homogeneous (such as cells used indrug discovery tests or multipotent stem cells) or cells that areexpensive to culture due to precise control of the differentiation state(specifically, stem cells such as iPS cells, ES cells, mesenchymal stemcells, cancer stem cells, and differentiation induction cells thereof;mesenchymal cells such as vascular endothelial cells, adipocytes,adipose stem cells, fibroblasts, cardiomyocytes, and myoblasts;epithelial cells such as HepaRA, HepaRG, HepG2, and BxPC-3; and thelike). In the case of primary cells, it suffices to select adherentcells that form colonies, which a person skilled in the art canappropriately select.

When forming the droplet of cell suspension in the coated region in anexample of this manufacturing method, the cell suspension is preferablydripped so that the shape of the bottom is circular. This techniquefacilitates obtaining a cell structure with a well-defined spheroidalshape.

In an example manufacturing method of Embodiment (IV), the number ofcells included in the droplet is preferably 3.0×10⁵ cells/mL or less. Inthe step of seeding cells, cells may precipitate in an overlapped stateif the cell density is too high, leading to cells that do not come incontact with the temperature-responsive polymer or thetemperature-responsive polymer composition. In this case, the cells thatare not in contact with the polymer or the polymer composition mighthave harmful effects, such as fusing with other cells and therebychanging the properties of the other cells. If the number of cellsincluded in the droplet is 3.0×10⁵ cells/mL or less, the cells can becaused to precipitate on the coated region nearly as a single layer,allowing the cells to be cultured under suitable conditions.

The number of cells included in the droplet is preferably 1.0×10⁴cells/mL or more to produce a sufficiently large cell structure.

For the same reasons as above, the number of cells included in thedroplet is more preferably 1.0×10⁵ cells/mL to 3.0×10⁵ cells/mL andparticularly preferably 2.0×10⁵ cells/mL to 3.0×10⁵ cells/mL. The numberof cells can be adjusted appropriately by a person skilled in the artconsidering the form, size, and the like of the seeded cells. Theaforementioned number of cells refers to the number of living cells.

In an example of this manufacturing method, the diameter of the dropletis preferably 1 μm to 8 mm.

If the diameter of the droplet is less than 1 μm, the amount of mediumbecomes too small, which might lead to conditions unfavorable for cellculturing, such as the medium drying out.

If the diameter of the droplet exceeds 8 mm, the density of cellsdisposed on the polymer may become uneven when the cells precipitateonto the polymer or polymer composition in the droplet, which mightproduce spheroidal shapes at a plurality of locations (i.e. a pluralityof stress points become the nucleus of cell aggregation) and prevent thespheroid from becoming a true sphere.

For the same reasons as above, the diameter of the droplet is morepreferably 100 μm to 4 mm and particularly preferably 300 μm to 3 mm.

In an example manufacturing method of Embodiment (IV), the amount of thedroplet is preferably 0.5 μL to 50 μL, more preferably 1.0 μL to 40 μL,and particularly preferably 5.0 μL to 25 μL to facilitate production ofa cell structure with a well-defined shape.

In an example of this manufacturing method, one droplet or a pluralityof droplets may be formed in one coated region.

In particular, when forming a plurality of droplets in one coatedregion, the interval between droplets (nearest distance betweendroplets) is preferably 1 μm to 500 μm.

If the distance is less than 1 μm, the droplets may combine with eachother. A distance exceeding 500 μm might prevent achievement ofefficient mass production of spheroids.

For the same reasons as above, the interval between droplets is morepreferably 2 μm to 250 μm and particularly preferably 5 μm to 100 μm.

In an example of this manufacturing method, cells are cultured in thedroplet (see (v) of FIG. 32).

The culture conditions may be determined appropriately in accordancewith the cells being used. An example is 37° C. and a 5% CO₂ atmosphere.

When culturing cells, a method of actively adding humidity to theculture container by spraying a mist of water or a method of inhibitingvolatilization of moisture in the droplet by covering the dropletsurface of the cell suspension with a film of an inert oil componentwith low specific gravity such as a phospholipid, a higher carboxylicacid, or liquid paraffin may be used. This method is particularlyeffective for maintaining the droplet of cell suspension when the amountof the droplet is less than 10 μL.

Subsequently, in an example of this manufacturing method, culturing isfurther continued to form a cell structure (see (vi) of FIG. 32).

The culture conditions may be determined appropriately in accordancewith the cells being used. An example is 37° C. and a 5% CO₂ atmosphere.

At this time, a cell structure that has an aggregated (pellet-like)structure can easily be formed in the coated region that has particularcharacteristics.

The following describes a preferred culturing surface used in amanufacturing method of a cell structure in an embodiment of Aspect(IV).

The preferred culturing surface includes a glass culturing surface andthe temperature-responsive polymer used in the above-describedmanufacturing method of a cell structure that starts at the culturingsurface in an embodiment of Aspect (IV).

In the preferred culturing surface, the glass itself provides resistanceto an organic solvent, allowing the culturing surface to be washed withan organic solvent and allowing a further reduction of cytotoxicsubstances. The culturing surface can also be manufactured without usinga conventional graft polymerization method that involves irradiation (asdescribed below), can reduce the amount of polymer decompositionproducts produced by irradiation, and can provide a good environment forcell growth.

Examples of a glass culturing surface include a culturing surface of aglass cell culture container and the surface of a glass plate.

Examples of temperature-responsive polymers include (A) atemperature-responsive polymer containing 2-N,N-dimethylaminoethylmethacrylate (DMAEMA) units and anionic monomer units and (B) atemperature-responsive polymer containing N-isopropyl acrylamide (NIPAM)units, cationic monomer units, and anionic monomer units, as describedabove.

In greater detail, examples of this preferred manufacturing method of acell culture container include (a) treating a glass culturing surfacewith a silane coupling agent having a vinyl group and then polymerizing2-N,N-dimethylaminoethyl methacrylate (DMAEMA) on the treated culturingsurface while irradiating with ultraviolet light in the presence ofwater, and (b) treating a glass culturing surface with a silane couplingagent having a halogenated alkyl group or introducing a N,N-dialkylsubstituted dithiocarbamoyl group by a substitution reaction of ahalogenated alkyl group and N,N-dialkyl substituted dithiocarbamyl acidand then subjecting 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) toiniferter polymerization, with the N,N-dialkyl substituteddithiocarbamoyl group as the starting point of radical polymerization,on the culturing surface while irradiating with ultraviolet light in thepresence of water.

In method (a), examples of the silane coupling agent having a vinylgroup include an agent having at least one selected from the groupconsisting of a vinyl group, a styryl group, a methacryl group, and anacryl group as a reactive functional group (Y) provided with thefunction of reacting or interacting with organic matter, and at leastone selected from the group consisting of an alkoxy group and a halogenas a reactive functional group (X) provided with the function ofreacting or interacting with glass.

Specific examples include vinyltrimethoxysilane,p-styryltrimethoxysilane, 3-methacryloyloxypropyltrimethoxysilane, and3-acryloyloxypropyltriethoxysilane.

Treatment with a silane coupling agent may be performed under normalconditions.

The method of polymerizing DMAEMA on a treated culturing surface may beas listed above regarding (A) a temperature-responsive polymercontaining 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) units andanionic monomer units.

In particular, the following conditions are preferably adopted in method(a).

The wavelength of ultraviolet light during ultraviolet light irradiationis preferably a wavelength of near ultraviolet light, more preferably330 nm to 400 nm, and particularly preferably 350 nm to 390 nm, toreduce the amount of polymer decomposition products.

Light is preferably irradiated from the back side of the treatedculturing surface (irradiated to pass through the glass), as doing socan cut the short wavelength ultraviolet light included in the lightsource to selectively irradiate light with a wavelength in the nearultraviolet region and can irradiate light from the side of the silanecoupling agent having a vinyl group towards the DMAEMA side toprioritize a polymerization reaction over monomer decomposition or thelike.

In method (b), examples of the silane coupling agent having ahalogenated alkyl group include an agent having a halogenated alkylgroup, specifically at least one selected from the group consisting of ap-chloromethylbenzyl group, a 3-chloropropyl group, ap-bromomethylbenzyl group, and a 3-bromopropyl group, as a reactivefunctional group (Y) provided with the function of reacting orinteracting with organic matter, and at least one selected from thegroup consisting of an alkoxy group and a halogen as a reactivefunctional group (X) provided with the function of reacting orinteracting with glass.

Specific examples include 3-chloropropyltrimethoxysilane,p-chloromethylbenzyltrichlorosilane, and 3-chloropropyltrichlorosilane.Treatment with a silane coupling agent may be performed under normalconditions.

Iniferter polymerization of the 2-N,N-dimethylaminoethyl methacrylate(DMAEMA), having an N,N-dialkyl substituted dithiocarbamoyl group as thestarting point of radical polymerization, introduced into the glasssurface (culturing surface) may be performed under normal reactionconditions.

In particular, the following conditions are preferably adopted in theaforementioned method of (b).

The wavelength of ultraviolet light during ultraviolet light irradiationis preferably a wavelength of near ultraviolet light, more preferably330 nm to 400 nm, and particularly preferably 350 nm to 390 nm, toreduce the amount of polymer decomposition products.

Light is preferably irradiated from the back side of the treatedculturing surface (irradiated to pass through the glass), as doing socan cut the short wavelength ultraviolet light included in the lightsource to selectively irradiate light with a wavelength in the nearultraviolet region and can irradiate light from the silane couplingagent side towards the DMAEMA side to prioritize a polymerizationreaction over monomer decomposition or the like.

An embodiment of a manufacturing method of a cell structure of Aspect(IV) has been described by examples with reference to the drawings, butthe manufacturing method of a cell structure of Aspect (IV) is notlimited to the above examples. Embodiment (IV) may be modified asappropriate.

[[Aspect (V)]]

With reference to the drawings, embodiments of a manufacturing method ofa cell structure of Aspect (V), a cell structure of Aspect (V), and acell culture container of Aspect (V) are described below in detail withexamples.

A manufacturing method of a cell structure of an embodiment of Aspect(V) (Embodiment (V)) includes:

a preparation step of preparing, on a culturing surface of a cellculture container, a first coated region coated with atemperature-responsive polymer and/or a temperature-responsive polymercomposition and a plurality of second coated regions located at the edgeof the first coated region and coated with a cell adhesive substance;and

a seeding and culturing step of seeding cells in the first coated regionand the second coated regions and culturing the cells.

An example manufacturing method in Embodiment (V) includes a productionstep of producing a temperature-responsive polymer and/or atemperature-responsive polymer composition, the above-describedpreparation step, and the above-described seeding and culturing step.

FIG. 35 is an overview, in (i) to (viii), of an example manufacturingmethod of a cell structure in Embodiment (V).

Details of each step in an example manufacturing method of a cellstructure in Embodiment (V) are provided below.

(Production Step)

Examples of the production step in Embodiment (V) include a step similarto the production step in Aspect (I), and a similar step is preferred.

(Preparation Step)

In an example manufacturing method, a first coated region coated with atemperature-responsive polymer and/or a temperature-responsive polymercomposition and a plurality of second coated regions located at the edgeof the first coated region and coated with a cell adhesive substance areprepared on a culturing surface of a cell culture container (preparationstep) (see (i) to (iii) of FIG. 35).

Here, the culturing surface apart from the first coated region and thesecond coated regions may be either cell adhesive or cell non-adhesive,but a cell non-adhesive culturing surface is preferable to facilitateobtaining a cell structure of the desired shape.

The production method of the cell non-adhesive culturing surface is notrestricted. For example, a cell culture container provided with a cellnon-adhesive culturing surface, such as PrimeSurface® by SUMILON, or acell non-adhesive sheet, pad, or the like may be used. A cell-culturecontainer provided with an untreated polystyrene culturing surface mayalso be used.

Here, as illustrated in FIG. 35, the first coated region and the secondcoated regions are preferably provided to be surrounded by thenon-coated region (the region of the culturing surface that is notparticularly coated) to suppress contact with the wall of the cellculture container and to adjust the shape of the cell structure (see(ii), (iii) of FIG. 35).

The area of the first coated region is not restricted. When, forexample, using a culturing surface of a ϕ35 mm cell culture container tomanufacture a cell structure with a size of 1 mm to 30 mm, the area ofthe first coated region may be 1 mm² to 750 mm², preferably 10 mm² to700 mm².

The shape of the first coated region is not restricted and may, in planview, be circular (a circle, an ellipse, or the like), polygonal (asquare, a rectangle, a triangle, or the like), or linear, and thecorners of a polygon may be rounded.

Among these shapes, a shape extending in a predetermined direction ispreferred to control the aggregation mode of cells. Specifically, ashape with a long axis and a short axis and a shape having a maximum andminimum distance between two points on the outer contour line arepreferable, and a rectangle is more preferable.

When the first coated region is a rectangle, the aspect ratio thereof(length of long sides:length of short sides) may be 1 to 50, preferably5 to 50, and more preferably 10 to 50.

When the first coated region is a rectangle, the width thereof ispreferably 0.1 mm to 50 mm, more preferably 0.1 mm to 30 mm, and thelength thereof is preferably 0.1 mm to 150 mm, more preferably 0.1 mm to100 mm, to control the shape of the cell structure.

The zeta potential of the surface of the first coated region ispreferably 0 mV to 50 mV, more preferably 0 mV to 35 mV, and even morepreferably 10 mV to 25 mV. A zeta potential of 0 mV or more facilitatesadhesion of negatively charged cells. A zeta potential of 50 mV or lesscan reduce cytotoxicity.

Furthermore, setting the zeta potential in the aforementioned rangesfurther facilitates production of cell structures having an aggregated(pellet-like) shape by simply culturing cells under appropriate cultureconditions. The reason is that setting the surface zeta potential withinthe aforementioned ranges is inferred to provide the surface of thefirst coated region with a weak positive charge that does not triggercytotoxicity, to ensure rapid adhesion of the seeded cells, to improvecell activity and encourage secretion of extracellular matrix, and alsoto appropriately inhibit cell migration, strengthening the bond betweencells.

The zeta potential refers to the value calculated with the Smoluchowskiequation by measurement using a zeta potential meter (for example, model“ELSZ” by Otsuka Electronics Co.) with a particle (zeta potential: −5 mVto +5 mV) in which polystyrene latex is coated with hydroxypropylcellulose as a reference monitor particle.

The contact angle of water relative to the surface of the first coatedregion is preferably 50° to 90°, more preferably 60° to 80°, and evenmore preferably 62° to 78° to increase the effects of Aspect (V). Thecontact angle of water relative to the first coated region refers to theaverage contact angle measured in conformity with JIS R3257 at severalarbitrary points within the coated region.

Examples of the cell adhesive substance coating the second coatedregions include laminin, collagen, fibronectin, peptides, cationicpolymers, and polystyrene. Examples of the peptides include peptidescontaining an arginine-glycine-aspartic acid sequence and peptidescontaining a sequence of 8 or more consecutive arginine residual groups.Examples of the cationic polymers include aminostyrene. Among these,laminin, collagen, and fibronectin, which have high cell adhesiveness,are preferable.

Reagents containing the above-listed cell adhesive substances can alsobe suitably used. Examples of such reagents include serum.

One type of these cell adhesive substances may be used alone, or acombination of two or more types may be used.

The area of the second coated region is not restricted. When, forexample, using a culturing surface of a ϕ35 mm cell culture container tomanufacture a cell structure with a size of 1 mm to 30 mm, the area ofthe second coated region may be 0.1 mm² to 75 mm², preferably 0.1 mm² to10 mm².

The shape of the second coated region is not restricted and may, in planview, be circular (a circle, an ellipse, or the like) or polygonal (asquare, a rectangle, a triangle, or the like), and the corners of apolygon may be rounded.

Among these shapes, a circular shape is preferred to lessen the forcethat acts on cells adhered to the second coated region when cellsaggregate.

When the second coated region is circular, the diameter thereof ispreferably 0.1 mm to 50 mm, more preferably 0.1 mm to 10 mm, to controlthe shape of the cell structure.

The ratio (S2/S1) of the area of the second coated region (S2) to thearea of the first coated region (S1) is not restricted but is preferably0.001 to 1.0, more preferably 0.01 to 0.5, to facilitate controlling theaggregation mode of cells.

In Embodiment (V), the first coated region and the second coated regionsmay overlap, the outer contour lines thereof may touch, or the regionsmay be separated by a shortest distance of 0.1 mm to 10 mm.

The position of the first coated region on the culturing surface and thepositions of the second coated regions on the culturing surface may bethe center of gravity of each region.

In Embodiment (V), it suffices for the second coated regions to beprovided at the edge of the first coated region. Here, the edge refersto the region 0.01 mm to 1 mm inward from the outer contour line of thefirst coated region.

In Embodiment (V), the number of second coated regions provided at theedge of the first coated region may be any number equal to or greaterthan 2, such as 3 or more, 4 or more, etc. The second coated regions maybe coated with the same cell adhesive substance or with differentsubstances.

FIGS. 36A to 36C illustrate arrangements of the first coated region andthe first coated region in Embodiment (V).

In an example illustrated in (i) of FIG. 36A and (i) to (viii) of FIG.35, the first coated region is circular in plan view, and the secondcoated regions are arranged to overlap the circular first coated regionat either side of the first coated region.

In Embodiment (V), the second coated region may be arranged at 3locations (see (ii) of FIG. 36A) or 4 locations (see (iii) of FIG. 36A)in overlap with the circular first coated region at the edge of thefirst coated region.

In Embodiment (V), the first coated region may be a rectangle withrounded corners, and the second coated regions may be arranged at eitheredge of the first coated region in overlap, as illustrated in FIG. 36B.

Furthermore, in Embodiment (V), the first coated region may have anydesired shape, and the second coated regions may be arranged at aplurality (5 in FIG. 36C) of edges of the first coated region inoverlap, as illustrated in FIG. 36C.

In an example manufacturing method of a cell structure illustrated inFIG. 35, the preparation step is performed by applying thetemperature-responsive polymer and/or the temperature-responsive polymercomposition to the central portion of the culturing surface of a cellculture container (see (i) of FIG. 35) while drawing a circular shape inplan view (see (ii) of FIG. 35) and then drying the applied region (see(iii) of FIG. 35) to provide the first coated region, and subsequentlyapplying a cell adhesive substance to two locations at the edge of thefirst coated region along a straight line passing through the center ofthe circular first coated region while drawing a circular shape in planview (see (ii) of FIG. 35) and then drying the applied region (see (iii)of FIG. 35) to provide the second coated regions.

The preparation step may, for example, be a step of dissolving atemperature-responsive polymer or a temperature-responsive polymercomposition in a solvent to form a solution including atemperature-responsive polymer or a temperature-responsive polymercomposition (temperature-responsive polymer solution), applying thesolution onto the culturing surface of a cell culture container, anddrying to prepare a coated cell culture container (preparation step I).The preparation step may also be a step of cooling an aqueous solutionincluding a temperature-responsive polymer or a temperature-responsivepolymer composition (temperature-responsive polymer aqueous solution) tothe cloud point of the temperature-responsive polymer or below, castingthe cooled temperature-responsive polymer aqueous solution onto theculturing surface of a cell culture container, and heating to atemperature above the cloud point to prepare a coated cell culturecontainer (preparation step II).

Examples of the solvent in the temperature-responsive polymer solutionin preparation step I include water; physiological saline; buffersolutions; alcohols such as methanol, ethanol, n-propyl alcohol,isopropyl alcohol, 1-butanol, isobutyl alcohol, 2-butanol, t-butylalcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol,2,2-dimethyl-1-propanol, 1-pentanol, 2-pentanol, 3-pentanol,2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol,2-methyl-2-pentanol, allyl alcohol, benzyl alcohol, and salicyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, methylpropyl ketone, methyl isobutyl ketone, methyl vinyl ketone,cyclohexanone, 2-methyl cyclopentanone, acetophenone, benzophenone, andisophorone; esters such as methyl acetate, ethyl acetate, propylacetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butylacetate, tert-butyl acetate, vinyl acetate, methyl formate, ethylformate, propyl formate, esters of the aforementioned alcohols andphosphoric acid, and esters of the aforementioned alcohols and carbonicacid; chloroform; benzene; toluene; diethyl ether; and dichloromethane.Among these, alcohols such as methanol, ethanol, n-propyl alcohol,isopropyl alcohol, 2-butanol, t-butyl alcohol, and allyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, and methylvinyl ketone; esters such as methyl acetate, ethyl acetate, isopropylacetate, tert-butyl acetate, and vinyl acetate; chloroform; benzene;toluene; diethyl ether; and dichloromethane are preferred forfacilitating uniform coating of the culturing surface and for havingexcellent solubility of temperature-responsive polymers. An organicsolvent with a low boiling point (such as at least one selected from thegroup consisting of a low-molecular alcohol with 1 to 4 carbon atoms, alow-molecular ketone with 3 to 5 carbon atoms, and an acetic acid alkylester having an alkyl group with 1 to 4 carbon atoms; in particular, atleast one selected from the group consisting of a low-molecular alcoholwith 1 to 4 carbon atoms, a low-molecular ketone with 3 to 5 carbonatoms, and an acetic acid alkyl ester having an alkyl group with 1 to 4carbon atoms, the low-molecular alcohol, low-molecular ketone, andacetic acid alkyl ester having a boiling point lower than that of water)is more preferable for allowing drying in a short time and facilitatingeven more uniform application on the culturing surface. Methanol andethanol are particularly preferable for their low cost and excellentoperability.

One type of these solvents may be used alone, or a combination of two ormore types may be used.

Since the solvent has excellent solubility with respect to atemperature-responsive polymer, the temperature-responsive polymer tendsnot to become insoluble and precipitate even at a temperature equal toor greater than the cloud point (such as room temperature or 37° C.).This eliminates the need to manage the temperature of thetemperature-responsive polymer during application of thetemperature-responsive polymer, allowing easy preparation of a coatedcell culture container.

In the preparation step I, a hydrophilic molecule is preferably includedin the temperature-responsive polymer to facilitate self-aggregation ofcells. Examples of the hydrophilic molecule include non-ionic,hydrophilic molecules that do not affect the C/A ratio of thetemperature-responsive polymer, such as polyethylene glycol (PEG),dimethyl acrylamide (DMAA), glycerin, Triton X, polypropylene glycol,and the like.

In the preparation step I, the content of the temperature-responsivepolymer in the temperature-responsive polymer solution is preferably0.00075 to 0.015 weight %, more preferably 0.001 to 0.01 weight %,relative to the temperature-responsive polymer solution (100 weight %)to facilitate uniform coating of the culturing surface by thetemperature-responsive polymer.

In the preparation step I, the content of the hydrophilic molecule inthe temperature-responsive polymer solution is preferably 0.00001 to0.00015 weight %, more preferably 0.00003 to 0.0001 weight %, relativeto the temperature-responsive polymer (100 weight %) to facilitateself-aggregation of cells.

To facilitate uniform coating of the culturing surface by thetemperature-responsive polymer or the temperature-responsive polymercomposition, the temperature-responsive polymer solution in thepreparation step I preferably does not include water, and the weightratio of water in the temperature-responsive polymer solution (100weight %) is more preferably 0.5 weight % or less and even morepreferably 0.1 weight % or less. The weight ratio of water can bemeasured by a method known to a person skilled in the art, such as gaschromatography or the Karl Fischer method.

In the preparation step I, the temperature-responsive polymer solutionmay be applied to the entire culturing surface or to a portion of theculturing surface, but application to the entire culturing surface ispreferable to facilitate obtaining a cell structure.

In the preparation step I, preferred conditions for drying the appliedtemperature-responsive polymer solution are drying under atmosphericpressure at a temperature of 10° C. to 70° C. for 1 to 3,000 minutes touniformly coat the culturing surface with the temperature-responsivepolymer or the temperature-responsive polymer composition. Quick dryingof the applied temperature-responsive polymer solution facilitatesuniform coating on the culturing surface with an even distribution ofthe temperature-responsive polymer or the temperature-responsive polymercomposition.

The applied temperature-responsive polymer solution may, for example, bedried by letting the cell culture container stand in an incubator at 37°C.

In the preparation step II, examples of the solvent for dissolving thetemperature-responsive polymer or the temperature-responsive polymercomposition include water; physiological saline; and buffer solutionssuch as a phosphate buffer solution, phosphate buffered saline (PBS),and a tris buffer solution.

In the preparation step II, examples of the method of cooling thetemperature-responsive polymer aqueous solution include placing thetemperature-responsive polymer aqueous solution in a refrigerator atapproximately 4° C. and cooling to a temperature at or below the cloudpoint.

In the preparation step II, examples of the method of casting thetemperature-responsive polymer aqueous solution onto the culturingsurface include a method of tilting the culturing surface of the cellculture container to spread the temperature-responsive polymer aqueoussolution that has a temperature at or below the cloud point and a methodof spreading the temperature-responsive polymer aqueous solution using aspatula.

In the preparation step II, examples of the method of heating the casttemperature-responsive polymer aqueous solution to above the cloud pointinclude a method of letting the cell culture container after the castingstep stand in an incubator at 37° C.

In the cell culture method of Embodiment (V), the region occupied by thefirst coated region and the second coated regions in the preparationstep is preferably surrounded by a cell non-adhesive wall. Specifically,the following modifications may be adopted.

Cell non-adhesive refers to adherent cells not adhering or tending notto adhere.

This embodiment inhibits adhesion between the seeded cells and the wallsof the recess and facilitates control of the aggregation form of cellsduring the below-described seeding and culturing step and allows moreprecise manufacturing of a cell structure having a desired orientation.

FIG. 37 is an outline of a modification to the preparation step, withthe subsequent seeding and culturing step.

In this modification to the preparation step, a recess with a planarshape forming the first coated region and the second coated regions inFIG. 35 (i.e. a large circle and two small circles arranged at the edgethereof) is carved into the culturing surface of a cell culturecontainer (see FIG. 37).

In the preparation step of this modification, the temperature-responsivepolymer and/or temperature-responsive polymer composition, along withthe cell adhesive substance, are arranged only on the bottom of thecarved-out recess (see (i) of FIG. 37). The polymer and/or polymercomposition, along with the cell adhesive substance, are not arranged onthe surface other than the bottom of the recess, i.e. the surface of thewalls of the recess.

This modification allows a cell structure having a desired orientationto be manufactured more precisely (see (ii) of FIG. 37).

(Seeding and Culturing Step)

In an example manufacturing method of Embodiment (V), cells are nextseeded in the first coated region and the second coated regions and arecultured to produce a cell structure (seeding and culturing step).

In an example in FIG. 35, the seeding and culturing step is performed byadding cells and a cell culture medium to the cell culture container(see (iv) of FIG. 35), subsequently placing the cell culture containerin a typical 37° C. cell incubator (see (v) of FIG. 35), adding new cellculture medium by medium exchange (see (vi) of FIG. 35), and placing thecell culture container in the cell incubator again (see (vii) and (viii)of FIG. 35).

Examples of the cells that can be used in the cell culturing method ofEmbodiment (V) include ADSC, cardiomyocytes, myocardial fibroblasts,nerve cells, neuroblasts, fibroblasts, chondrocytes, vascularendothelial cells, vascular stromal cells, and smooth muscle cells.

One type of these cells may be used alone, or a combination of two ormore types may be used.

The cell density when seeding cells in the seeding and culturing step ispreferably 0.3×10⁴ cells/cm² or more, and more preferably 1.0×10⁴cells/cm² or more. Furthermore, to prevent problems related to the cellcycle, such as growth arrest due to contact between cells duringculturing, the cell density is preferably 5.0×10⁴ cells/cm² or less andmore preferably 4.5×10⁴ cells/cm² or less.

The first coated region after seeding preferably has a confluency of 90%to 100%, more preferably 95% to 100%, and even more preferably 99% to100%.

Setting the confluency within the aforementioned ranges prevents thecells from each forming colonies, allowing the cells to aggregate andform a cell structure while remaining homogeneously dispersed.Furthermore, the seeded cells do not grow easily if the cell density ishigh, allowing formation of an aggregated cell structure before cellsgrow. Since the properties of seeded cells may change when the cellsgrow, impeding cell growth allows formation of a cell structureincluding cells with the same properties as at the time of seeding.

A person skilled in the art can appropriately determine the cultureconditions on the basis of the type of cells being used and the purposeof the experiment. Example conditions are 37° C. and a 5% CO₂atmosphere.

The phenomenon that occurs in the seeding and culturing step isdescribed below with reference to FIG. 35.

In this step, the seeded cells first precipitate on the first coatedregion and the second coated regions. At this time, the cells thatprecipitate on the first coated region and the second coated regionsadhere to the coated regions and survive, whereas the cells thatprecipitate on the non-coated region die without adhering to thenon-coated region (see (v) of FIG. 35).

Next, the cells that did not adhere to the culturing surface are removedfrom the cell culture container (see (vi) of FIG. 35).

Upon further culturing of the cells adhered to the first coated regionand the second coated regions, the cells located near the boundarybetween the first coated region and the non-coated region start toaggregate towards the central portion of the first coated region, alongwith the cells positioned closer to the central portion of the firstcoated region (see (vii) of FIG. 35). In other words, the adhered cellspeel away from the first coated region, warping towards the centralportion of the first coated region.

On the other hand, the cells adhered to the second coated regions remainadhered to their initial position, without peeling off from the secondcoated regions (see (vii) of FIG. 35).

Ultimately, the cells adhered to the first coated region form a cellstructure with a linear structure at the central portion of the firstcoated region, connecting the two cell groups that remain adhered to thesecond coated regions (see (viii) of FIG. 35).

Here, within the cell structure obtained in the seeding and culturingstep, the cells in the portion formed by cells that were adhered to thefirst coated region are stretched in the extending direction of thelinear structure and are oriented along a line connecting the two secondcoated regions.

A plurality of cell structures obtained in this way can be interwoven orconnected appropriately to produce a cell structure with a desiredshape.

In terms of vitality, the resulting cell structure is preferably usedimmediately after formation, insofar as possible, in an experiment orthe like and is more preferably used within 24 hours.

In the manufacturing method of a cell structure of Embodiment (V),single layer cells supplied with sufficient nutrients and oxygenaggregate in a short period of time to form a cell structure, therebyavoiding problems with known techniques such as cells on the inside ofthe cell structure dying from lack of oxygen. This method isparticularly useful for culturing a cell structure composed ofcardiomyocytes, which have a particularly strong need for oxygen.

Obtaining, in a test tube, a cell structure within the same environmentas a living organism is extremely important in terms of experimentaltechnique. The manufacturing method of a cell structure of Embodiment(V) allows the orientation of cells to be matched to the orientation ina living organism (for example, a spindle form in a predetermineddirection in the case of cardiomyocytes).

For example, during drug discovery tests related to cardiomyocytes, theelectrocardiographic waveform of cultured cardiomyocytes is evaluated todetect cardiotoxicity, such as arrhythmogenesis. To increase themeasurement accuracy (S/N ratio), the electric signal from the cells ispreferably as large as possible.

As described above, the manufacturing method of a cell structure ofEmbodiment (V) allows a large cell structure in a state with theoriginal orientation to be obtained. Hence, a specimen imitating livingcardiomyocytes in a living organism can be obtained. This can improvethe accuracy of drug discovery tests related to cardiomyocytes.

[[Aspect (VI)]]

[Manufacturing Method of a Cell Structure]

A manufacturing method of a cell structure of Aspect (VI) includes thesuccessive steps of a production step of producing atemperature-responsive polymer or a temperature-responsive polymercomposition, a culture container preparation step of coating a culturingsurface of a cell culture container with the temperature-responsivepolymer or the temperature-responsive polymer composition to prepare acoated cell culture container, a seeding step of seeding cardiomyocytesand fibroblasts in the coated cell culture container at a ratio of 200to 300 fibroblasts per 100 cardiomyocytes, and a culturing step ofculturing the seeded cells to obtain an aggregated cell structure.

In the present disclosure, the coated portion, on the culturing surfaceof the cell culture container, coated with the temperature-responsivepolymer or the temperature-responsive polymer composition is alsoreferred to as a “coated culturing surface”. The coated cell culturecontainer is a cell culture container with the coated culturing surface.The entire culturing surface of the coated cell culture container may bethe coated culturing surface, or a portion of the culturing surface maybe the coated culturing surface. The culturing surface may have onecoated culturing surface or a plurality of coated culturing surfaces.

A cell structure including cardiomyocytes and fibroblasts can be formedinto a spheroid over a long time of 1 to 2 weeks using a well-knownU-shaped low-adhesion culture dish or the hanging drop method.Production is time consuming, however, leading to problems such asdifficulty maintaining cardiomyocytes, which are extremely susceptibleto a hypoxic state, and difficulty coculturing cells with differentgrowth rates.

We discovered the surprising fact that seeding cardiomyocytes andfibroblasts in a coated cell culture container in which the culturingsurface is coated with a temperature-responsive polymer or atemperature-responsive polymer composition allows easy formation of acell structure imitating heart disease tissue that has suffered cardiacfailure.

The manufacturing method of a cell structure of Embodiment (VI) allowscardiomyocytes and fibroblasts to aggregate while maintaining a state ofhomogeneous dispersion and allows reproduction of tissue, in a stateclose to a state of existence in a living organism, that includescardiomyocytes and fibroblasts and that has suffered cardiac failure.

In the manufacturing method of a cell structure of Embodiment (VI), theseeded mixed cells adhere to the coated culturing surface, and uponbeing cultured, self-aggregate and contract so that the edge of thecells that are spread out like a sheet separates from the coatedculturing surface and warps. The cells thus aggregate to form anaggregated cell structure.

In particular, in the manufacturing method of a cell structure ofEmbodiment (VI), the culturing surface is coated by atemperature-responsive polymer or a temperature-responsive polymercomposition, and the adhesiveness between the coated culturing surfaceand the cells is in an appropriate range. Therefore, even mixed cellsincluding cardiomyocytes, which do not readily aggregate, can form anaggregated cell structure. An aggregated cell structure can also beformed easily by a combination with fibroblasts, which readilyaggregate.

Since cardiomyocytes and fibroblasts have different growth rates, aknown method of forming a cell structure by culturing for a long timedoes not allow formation of a cell structure that includes manycardiomyocytes. The manufacturing method of a cell structure ofEmbodiment (VI) allows formation of a cell structure without growingcardiomyocytes and fibroblasts, thereby yielding a cell structureconstituted by cells in nearly the same ratio as the cell ratio betweencardiomyocytes and fibroblasts at the time of seeding. The mixing ratioof cells constituting the cell structure can therefore easily beadjusted. Furthermore, the manufacturing method of a cell structure ofEmbodiment (VI) uses a coated cell culture container in which theadhesiveness between the coated culturing surface and the cells is in anappropriate range and therefore does not require cells to be grown. Forexample, a mixture of cardiomyocytes and fibroblasts aggregates androlls up in a short period of time, such as within 24 hours afterseeding, thereby easily allowing formation of an aggregated cellstructure that is constituted by two or more types of cells withdifferent growth rates, that includes living cardiomyocytes, and that isbeating.

(Production Step)

Examples of the production step of producing the temperature-responsivepolymer and the temperature-responsive polymer composition used in acell culture container of Embodiment (VI) include a step similar to theproduction step in Aspect (I), and a similar step is preferred.

In Embodiment (VI), the temperature-responsive polymer and thetemperature-responsive polymer composition used in the manufacturingmethod are preferably (A) to further facilitate obtaining an aggregatedcell structure that includes cardiomyocytes and fibroblasts.

The production step of producing a mixture containing2-N,N-dimethylaminoethyl methacrylate (DMAEMA) in Embodiment (VI) isalso referred to as a mixture production step.

(Culture Container Preparation Step)

In the manufacturing method of an embodiment of Aspect (VI), the culturecontainer preparation step is a step of coating a culturing surface of acell culture container with the temperature-responsive polymer or thetemperature-responsive polymer composition to prepare a coated cellculture container.

The culture container preparation step may, for example, be a step ofdissolving a temperature-responsive polymer or a temperature-responsivepolymer composition in a solvent to form a temperature-responsivepolymer solution, applying the solution onto the culturing surface of acell culture container, and drying to prepare a coated cell culturecontainer (culture container preparation step I). The culture containerpreparation step may instead be a step of cooling an aqueous solutionincluding a temperature-responsive polymer or a temperature-responsivepolymer composition (temperature-responsive polymer aqueous solution) tothe cloud point of the temperature-responsive polymer or below, castingthe cooled temperature-responsive polymer aqueous solution onto theculturing surface of a cell culture container, and heating to atemperature above the cloud point to prepare a coated cell culturecontainer (culture container preparation step II).

Examples of the solvent in the temperature-responsive polymer solutionin the culture container preparation step I include water; physiologicalsaline; buffer solutions; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 1-butanol, isobutyl alcohol, 2-butanol,t-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol,2,2-dimethyl-1-propanol, 1-pentanol, 2-pentanol, 3-pentanol,2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol,2-methyl-2-pentanol, allyl alcohol, benzyl alcohol, and salicyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, methylpropyl ketone, methyl isobutyl ketone, methyl vinyl ketone,cyclohexanone, 2-methyl cyclopentanone, acetophenone, benzophenone, andisophorone; esters such as methyl acetate, ethyl acetate, propylacetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butylacetate, tert-butyl acetate, vinyl acetate, methyl formate, ethylformate, propyl formate, esters of the aforementioned alcohols andphosphoric acid, and esters of the aforementioned alcohols and carbonicacid; chloroform; benzene; toluene; diethyl ether; and dichloromethane.

Among these, water; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 2-butanol, t-butyl alcohol, and allylalcohol; ketones such as acetone, ethyl methyl ketone, diethyl ketone,and methyl vinyl ketone; esters such as methyl acetate, ethyl acetate,isopropyl acetate, tert-butyl acetate, and vinyl acetate; chloroform;benzene; toluene; diethyl ether; and dichloromethane are preferred forfacilitating uniform coating of the culturing surface and for havingexcellent solubility of temperature-responsive polymers. An organicsolvent with a low boiling point (such as at least one selected from thegroup consisting of a low-molecular alcohol with 1 to 4 carbon atoms, alow-molecular ketone with 3 to 5 carbon atoms, and an acetic acid alkylester having an alkyl group with 1 to 4 carbon atoms; in particular, atleast one selected from the group consisting of a low-molecular alcoholwith 1 to 4 carbon atoms, a low-molecular ketone with 3 to 5 carbonatoms, and an acetic acid alkyl ester having an alkyl group with 1 to 4carbon atoms, the low-molecular alcohol, low-molecular ketone, andacetic acid alkyl ester having a boiling point lower than that of water)is more preferable for allowing drying in a short time and facilitatingeven more uniform application on the culturing surface. Methanol andethanol are particularly preferable for their low cost and excellentoperability.

One type of these solvents may be used alone, or a combination of two ormore types may be used.

Since the solvent has excellent solubility with respect to atemperature-responsive polymer, the temperature-responsive polymer tendsnot to become insoluble and precipitate even at a temperature equal toor greater than the cloud point (such as room temperature or 37° C.).This eliminates the need to manage the temperature of thetemperature-responsive polymer during application of thetemperature-responsive polymer, allowing easy preparation of a coatedcell culture container.

In the culture container preparation step I, a hydrophilic molecule ispreferably included in the temperature-responsive polymer to facilitateself-aggregation of cells. Examples of the hydrophilic molecule includenon-ionic, hydrophilic molecules that do not affect the C/A ratio of thetemperature-responsive polymer, such as polyethylene glycol (PEG),dimethyl acrylamide (DMAA), glycerin, Triton X, polypropylene glycol,and the like.

In the culture container preparation step I, the content of thetemperature-responsive polymer in the temperature-responsive polymersolution is preferably 0.05 mass % to 2.0 mass %, more preferably 0.1mass % to 1.5 mass %, relative to the temperature-responsive polymersolution (100 mass %) to facilitate uniform coating of the culturingsurface by the temperature-responsive polymer.

In the culture container preparation step I, the content of thehydrophilic molecule in the temperature-responsive polymer solution ispreferably 0.00001 mass % to 0.00015 mass %, more preferably 0.00003mass % to 0.0001 mass %, relative to the temperature-responsive polymer(100 mass %) to facilitate self-aggregation of cells.

To facilitate uniform coating of the culturing surface by thetemperature-responsive polymer or the temperature-responsive polymercomposition, the temperature-responsive polymer solution in the culturecontainer preparation step I preferably does not include water, and themass ratio of water in the temperature-responsive polymer solution (100mass %) is more preferably 0.5 mass % or less and even more preferably0.1 mass % or less.

The mass ratio of water can be measured by a method known to a personskilled in the art, such as gas chromatography or the Karl Fischermethod.

In the culture container preparation step I, the temperature-responsivepolymer solution may be applied to the entire culturing surface or to aportion of the culturing surface. When the temperature-responsivepolymer solution is applied to a portion of the culturing surface, onecoated culturing surface or a plurality of coated culturing surfaces maybe provided on the culturing surface. A cell culture container with acell non-adhesive culturing surface is preferably used when thetemperature-responsive polymer solution is applied to a portion of theculturing surface.

“Cell non-adhesive” refers to adherent cells (for example, fibroblasts,cardiomyocytes, vascular endothelial cells, and the like) either notadhering or tending not to adhere under normal culture conditions.

In the culture container preparation step I, preferred conditions fordrying the applied temperature-responsive polymer solution are dryingunder atmospheric pressure at a temperature of 10° C. to 70° C. for 1 to3,000 minutes to uniformly coat the culturing surface with thetemperature-responsive polymer or the temperature-responsive polymercomposition. Quick drying of the applied temperature-responsive polymersolution facilitates uniform coating on the culturing surface with aneven distribution of the temperature-responsive polymer or thetemperature-responsive polymer composition.

The applied temperature-responsive polymer solution may, for example, bedried by letting the cell culture container stand in an incubator at 37°C.

In the culture container preparation step II, examples of the solventfor dissolving the temperature-responsive polymer or thetemperature-responsive polymer composition include water; physiologicalsaline; and buffer solutions such as a phosphate buffer solution,phosphate buffered saline (PBS), and a tris buffer solution.

In the culture container preparation step II, examples of the method ofcooling the temperature-responsive polymer aqueous solution includeplacing the temperature-responsive polymer aqueous solution in arefrigerator at approximately 4° C. and cooling to a temperature at orbelow the cloud point.

In the culture container preparation step II, examples of the method ofcasting the temperature-responsive polymer aqueous solution onto theculturing surface include a method of tilting the culturing surface ofthe cell culture container to spread the temperature-responsive polymeraqueous solution that has a temperature at or below the cloud point anda method of spreading the temperature-responsive polymer aqueoussolution using a spatula.

In the culture container preparation step II, examples of the method ofheating the cast temperature-responsive polymer aqueous solution toabove the cloud point include a method of letting the cell culturecontainer after the casting step stand in an incubator at 37° C.

Examples of the cell culture container include commercially availablemultiwell plates, dishes, flasks, and the like. Examples of the materialof the cell culture container include polystyrene, polyethyleneterephthalate (PET), polypropylene, polybutene, polyethylene,polycarbonate, and glass. Among these, polystyrene and polyethyleneterephthalate (PET) are preferable for being easy to mold precisely, forallowing adoption of various sterilization methods, and for beingsuitable for microscope observation by virtue of being transparent.

Cell adhesion treatment or the like may be applied to the culturingsurface of the cell culture container, or the surface may be untreated.The culturing surface may be coated, processed, or the like to adjustthe cell adhesiveness.

The planar shape of the culturing surface is not restricted and may, forexample, be a substantially rectangular shape or other substantiallypolygonal shape, a substantially circular shape, or the like. Amongthese, a substantially circular shape is preferred to facilitateobtaining a more homogeneous cell structure.

The bottom shape of the culturing surface (the cross-sectional shape ofthe bottom) is not restricted, and examples include a flat bottom, roundbottom, and uneven bottom. Among these, a flat bottom or a slightlycurved concave bottom is preferable to facilitate obtaining a morehomogeneous, spheroidal cell structure.

The area of the culturing surface of the cell culture container ispreferably 200 mm² or less, more preferably 75 mm² or less, and evenmore preferably 32 mm² or less, from the perspective of supplying oxygento the cardiomyocytes. The lower limit on the area of the culturingsurface of the cell culture container is not restricted, and anycommercially available size may be used.

The area of each coated culturing surface (the area of each portioncoated with a temperature-responsive polymer or a temperature-responsivepolymer composition) is preferably 200 mm² or less, more preferably 75mm² or less, and even more preferably 32 mm² or less, from theperspective of supplying oxygen to the cardiomyocytes.

In the coated cell culture container, the amount oftemperature-responsive polymer per unit area of the coated culturingsurface is preferably 0.1 ng/mm² to 10.0 ng/mm² and more preferably 0.5ng/mm² to 5.0 ng/mm². Adopting these ranges facilitates achievement ofthe effect of easier formation of an aggregated cell structure.

The zeta potential of the coated culturing surface in the coated cellculture container is preferably 0 mV to 50 mV, more preferably 0 mV to35 mV, and even more preferably 10 mV to 25 mV. A zeta potential of 0 mVor more facilitates adhesion of negatively charged cells. A zetapotential of 50 mV or less can reduce cytotoxicity.

Furthermore, setting the zeta potential in the aforementioned rangesfurther facilitates production of an aggregated (pellet-like) cellstructure by simply culturing cells under appropriate cultureconditions. The reason is that setting the surface zeta potential withinthe aforementioned ranges is inferred to provide the coated culturingsurface with a weak positive charge that does not trigger cytotoxicity,to ensure rapid adhesion of the seeded cells, to improve cell activityand encourage secretion of extracellular matrix, and also toappropriately inhibit cell migration, strengthening the bond betweencells. The zeta potential refers to the value calculated with theSmoluchowski equation by measurement using a zeta potential meter (forexample, model “ELSZ” by Otsuka Electronics Co.) with a particle (zetapotential: −5 mV to +5 mV) in which polystyrene latex is coated withhydroxypropyl cellulose as a reference monitor particle.

The contact angle of water relative to the coated culturing surface ispreferably 50° to 90°, more preferably 60° to 80°, and even morepreferably 620 to 780 to increase the effects of Aspect (VI). Thecontact angle of water relative to the coated culturing surface refersto the average contact angle measured in accordance with JIS R3257 atany number of points on the coated culturing surface.

(Seeding Step)

The seeding step is a step of seeding cardiomyocytes and fibroblasts inthe coated cell culture container at a ratio of 200 to 300 fibroblastsper 100 cardiomyocytes. The cells may all be mixed and seeded at once orseeded a portion at a time. Each type of cell may also be seeded at onceor seeded a portion at a time.

The cells seeded on the coated culturing surface of the coated cellculture container include at least cardiomyocytes and fibroblasts.

Cardiomyocytes, myocardial stem cells, and iPS cells, ES cells, or thelike in the differentiation induction stage are preferred as theaforementioned cardiomyocytes to facilitate obtaining a cell structurein which cardiomyocytes and fibroblasts are more homogenously dispersed.

One type of cardiomyocytes may be used alone, or a combination of two ormore types may be used.

Examples of the fibroblasts include myocardial fibroblasts, subcutaneousadipose, fascia, synovium, and periosteal-derived mesenchymal stemcells. Among these, myocardial fibroblasts and subcutaneousadipose-derived mesenchymal stem cells are preferred to facilitateobtaining a cell structure in which cardiomyocytes and fibroblasts aremore homogenously dispersed.

One type of fibroblasts may be used alone, or a combination of two ormore types may be used.

In the seeding step, vascular endothelial cells or vascular stromalcells may be also be seeded. One type of these cells may be used alone,or a combination of two or more types may be used.

In the seeding step, immune system cells may also be seeded.

Examples of the immune system cells include macrophages, T cells such asCD4+ T cells, and monocytes. Among these, inclusion of macrophagesand/or T cells is preferred to facilitate obtaining a cell structureeven closer to tissue that has suffered heart disease such as cardiacfailure.

One type of these immune system cells may be used alone, or acombination of two or more types may be used.

In the seeding step, the number of fibroblasts per 100 cardiomyocytes is200 to 300, preferably 200 to 250, and more preferably 200 to 230.Setting the number of fibroblasts per 100 cardiomyocytes to be 200 ormore facilitates curling of the mixed cells to form an aggregated cellstructure, and setting the number to be 300 or less allows a cellstructure closer to tissue that has suffered heart disease such ascardiac failure to be obtained.

In the seeding step, the ratio of cardiomyocytes among all of the seededcells is preferably 25% to 50% of all of the seeded cells (100%), morepreferably 25% to 30%, to obtain a cell structure closer to tissue thathas suffered heart disease such as cardiac failure.

In the seeding step, the number of vascular endothelial cells per 100cardiomyocytes is preferably 5 to 50, more preferably 10 to 30, toobtain a cell structure closer to tissue that has suffered heart diseasesuch as cardiac failure.

In the seeding step, the number of immune system cells per 100cardiomyocytes is preferably 5 to 50, more preferably 10 to 30, toobtain a cell structure closer to tissue that has suffered heart diseasesuch as cardiac failure.

In the seeding step, the density of all of the seeded cells on thecoated culturing surface is preferably a confluency of 90% to 100%relative to the surface area of the coated culturing surface, morepreferably a confluency of 95% to 100%, and even more preferably aconfluency of 99% to 100%. Setting the density of seeded cells withinthe aforementioned ranges prevents the cells from each forming colonies,allowing the cells to aggregate and form a cell structure whileremaining homogeneously dispersed. Furthermore, the seeded cells do notgrow easily if the cell density is high, allowing formation of anaggregated cell structure before cells grow. Hence, a cell structureincluding cells in the same ratio as the seeded cells can be formed. Thedifference in growth rates of cells also has little effect. Since theproperties of seeded cells may change when the cells grow, impeding cellgrowth allows formation of a cell structure including cells with thesame properties as at the time of seeding.

While the density of all of the seeded cells on the coated culturingsurface depends on the type of cell, a density of 20 cells/mm² to 15,000cells/mm² is preferred. For example, when seeding by adding 1.0 mL ofcell fluid to a 24 well cell culture plate with a coated culturingsurface area of 200 mm², the density is preferably 4×10⁴ cells/mL to30×10⁴ cells/mL. Live cells are seeded.

The coated cell culture container may be left to stand in an incubatorat 37° C. and subsequently removed and placed on a clean bench at roomtemperature, for example, with cell seeding then being performed.

Cells are preferably seeded after being diluted in a medium. The mediumfor dilution may be any medium in which cells can be cultured.

(Culturing Step)

The culturing step is a step of culturing the seeded cells to obtain anaggregated cell structure.

It is known that cardiomyocytes necrotize in tissue that has sufferedmyocarditis, the necrotic cardiomyocytes are replaced by excessivegrowth of fibroblasts, and moreover, many inflammatory immune cellsinfiltrate. To reproduce, in a test tube, myocarditis tissue into whichimmune system cells have infiltrated, it is desirable to produce a cellstructure that includes cardiomyocytes and fibroblasts and into whichimmune system cells, which are suspended cells, have infiltrated.However, this is difficult with a known hanging drop method orlow-adhesion culture dish, which require 1 to 2 weeks to produce cellstructures.

In the manufacturing method of Embodiment (VI), immune system cells maybe further added to the coated cell culture container in the culturingstep. The timing at which immune system cells are added is preferablyduring or after the aforementioned seeding step (simultaneous with orafter cell seeding) and before a cell structure is obtained. The timingis more preferably when or after seeded cells adhere to the coatedculturing surface and form a sheet-like cell structure (see (iii) ofFIG. 40) and before the sheet-like cell structure starts to aggregatetowards the central portion of the coated culturing surface and the edgeseparates from the coated culturing surface and starts to warp, forminga cell structure with a warped edge. In other words, the immune systemcells may be seeded during the seeding step or added after the seedingstep.

Specifically, the immune system cells are added simultaneous with orafter cell seeding, preferably within 48 hours after seeding of cells(for example, after the cells that are seeded last).

Adding the immune system cells at the aforementioned timing allows theimmune system cells, which are suspended cells, to precipitate due togravity. When cells adhered to the coated culturing surface aggregatelike a drawstring bag, the immune system cells are incorporated inside,allowing formation of a cell structure that includes immune system cellson the inside. A cell structure even closer to heart disease modeltissue that has suffered myocarditis, cardiac failure, or the like canthus be obtained (see FIG. 40).

The conditions for culturing the seeded cells may, for example, be theuse of a typical cell incubator at 37° C. The cells are preferablycultured continuously until forming an aggregated cell structure.Specifically, the cells are preferably cultured for 10 hours to 96 hoursand more preferably for 15 hours to 50 hours.

Surprisingly, even when mixing, seeding, and culturing adherent cells(such as cardiomyocytes) and suspended cells (such as immune systemcells), the manufacturing method of a cell structure of Embodiment (VI)allows a cell structure to be obtained by seeding and culturing theadherent cells and the suspended cells simultaneously using a mediumsuitable for the adherent cells being used or a medium suitable for thesuspended cells being used (preferably a medium suitable for theadherent cells), without the need for preparation of a special medium.

Mixed cells including the cardiomyocytes and the fibroblasts, whichadhere to the coated culturing surface and are cultured, self-aggregateto form an aggregated cell structure. The resulting cell structureincludes living cells inside the aggregated structure.

The cell structure preferably has, for example, an outer diameter of 30μm to 200 μm, more preferably 40 μm to 150 μm, and even more preferably50 μm to 100 μm, to obtain a beating cell structure that includes livingcardiomyocytes on the inside.

The cell structure obtainable with the manufacturing method of a cellstructure of Embodiment (VI) is a heart disease model reproducing tissuethat has suffered heart disease such as cardiac failure and can be usedfor purposes including research on heart disease, such as myocardialinfarction, cardiac failure, and myocarditis; development of drugs forheart disease; and development of treatment for heart disease.

An example manufacturing method of a cell structure of Embodiment (VI)is described below with reference to FIGS. 39 and 40.

FIG. 39 illustrates an example manufacturing method of a cell structurethat includes cardiomyocytes and fibroblasts.

A temperature-responsive polymer is applied to a culturing surface of acell culture container to coat the culturing surface, thereby preparinga coated cell culture container having a coated culturing surface (see(i) and (ii) of FIG. 39). Subsequently, mixed cells composed ofcardiomyocytes and fibroblasts and diluted with medium are added to thecoated culturing surface to seed the mixed cells (see (ii) of FIG. 39).The seeded mixed cells adhere to the entire coated culturing surface(see (iii) of FIG. 39). In this example, the density of the mixed cellsis 100% confluency (see (iii) of FIG. 39). Subsequently, the mixed cellsadhered to the coated culturing surface start to aggregate towards thecentral portion of the coated culturing surface, and the edge separatesfrom the coated culturing surface and starts to warp, yielding a cellstructure with a warped edge (see (iv) of FIG. 39). The cells thencontinue to aggregate to form an aggregated cell structure, which floatsfrom the coated culturing surface (see (v) of FIG. 39).

FIG. 40 illustrates an example manufacturing method of a cell structurethat includes cardiomyocytes, fibroblasts, and immune system cells(suspended cells) such as macrophages.

A temperature-responsive polymer is applied to a culturing surface of acell culture container to coat the culturing surface, thereby preparinga coated cell culture container having a coated culturing surface (see(i) and (ii) of FIG. 40). Subsequently, mixed cells composed ofcardiomyocytes and fibroblasts and diluted with medium are added to thecoated culturing surface to seed the mixed cells (see (ii) of FIG. 40).The seeded mixed cells adhere to the entire coated culturing surface toform a sheet-like (single layer) cell structure (see (iii) of FIG. 40).In this example, the density of the mixed cells is 100% confluency (see(iii) of FIG. 40).

After the cardiomyocytes and fibroblasts form the sheet-like cellstructure, immune system cells such as macrophages are added ((iv) ofFIG. 40). Subsequently, the mixed cells adhered to the coated culturingsurface start to aggregate towards the central portion of the coatedculturing surface, and the edge separates from the coated culturingsurface and starts to warp, yielding a cell structure with a warped edge(see (v) of FIG. 40). At this time, the cell structure incorporates theimmune system cells that are adhered to the sheet-like cell structure orare suspended in the culture dish. The cells then continue to aggregateto form an aggregated cell structure that includes cardiomyocytes,fibroblasts, and immune system cells. The cell structure then floatsfrom the coated culturing surface (see (vi) of FIG. 40).

[[Aspect (VII)]]

[Manufacturing Method of a Cell Structure]

A manufacturing method of a cell structure of Aspect (VII) includes thesuccessive steps of a production step of producing atemperature-responsive polymer or a temperature-responsive polymercomposition, a culture container preparation step of coating a culturingsurface of a cell culture container with the temperature-responsivepolymer or the temperature-responsive polymer composition to prepare acoated cell culture container, a seeding step of seeding hepatocytes andfibroblasts in a ratio of 10 to 50 fibroblasts per 100 hepatocytes inthe coated cell culture container, and a culturing step of culturing theseeded cells to obtain an aggregated cell structure.

In the present disclosure, the coated portion, on the culturing surfaceof the cell culture container, coated with the temperature-responsivepolymer or the temperature-responsive polymer composition is alsoreferred to as a “coated culturing surface”. The coated cell culturecontainer is a cell culture container with the coated culturing surface.The entire culturing surface of the coated cell culture container may bethe coated culturing surface, or a portion of the culturing surface maybe the coated culturing surface. The culturing surface may have onecoated culturing surface or a plurality of coated culturing surfaces.

A spheroidal cell structure including hepatocytes and fibroblasts can beformed over a long time of 1 to 2 weeks using a well-known U-shapedlow-adhesion culture dish or the hanging drop method. Production is timeconsuming, however, leading to problems such as difficulty coculturingcells whose growth rates, adhesiveness to the culturing surface, andsuitable culture conditions differ.

We discovered the surprising fact that seeding hepatocytes andfibroblasts in a coated cell culture container in which the culturingsurface is coated with a temperature-responsive polymer or atemperature-responsive polymer composition allows easy formation of acell structure imitating tissue that has suffered hepatic failure or thelike.

The manufacturing method of a cell structure of Embodiment (VII) allowshepatocytes and fibroblasts to aggregate while maintaining a state ofhomogeneous dispersion and allows reproduction of tissue, in a stateclose to a state of existence in a living organism, that includeshepatocytes and fibroblasts and that has suffered hepatic failure.

Since hepatocytes and fibroblasts have different growth rates,adhesiveness to the culturing surface, and suitable culture conditions,it is extremely difficult to form a cell structure including hepatocytesand fibroblasts with a known method of forming a cell structure byculturing for a long time. The manufacturing method of a cell structureof Embodiment (VII) uses a coated cell culture container in which theadhesiveness between the coated culturing surface and the cells is in anappropriate range and therefore does not require cells to be grown. Forexample, hepatocytes and fibroblasts aggregate and roll up in a shortperiod of time, such as within 24 hours after seeding, thereby easilyallowing formation of an aggregated cell structure that includeshepatocytes and fibroblasts.

(Production Step)

Examples of the production step of producing the temperature-responsivepolymer and the temperature-responsive polymer composition used in acell culture container of Embodiment (VII) include a step similar to theproduction step in Aspect (I), and a similar step is preferred.

In Embodiment (VII), the temperature-responsive polymer and thetemperature-responsive polymer composition used in the manufacturingmethod are preferably (A) to further facilitate obtaining an aggregatedcell structure that includes hepatocytes and fibroblasts.

The production step of producing a mixture containing2-N,N-dimethylaminoethyl methacrylate (DMAEMA) in Embodiment (VII) isalso referred to as a mixture production step.

(Culture Container Preparation Step)

In the manufacturing method of an embodiment of Aspect (VII), theculture container preparation step is a step of coating a culturingsurface of a cell culture container with the temperature-responsivepolymer or the temperature-responsive polymer composition to prepare acoated cell culture container.

The culture container preparation step may, for example, be a step ofdissolving a temperature-responsive polymer or a temperature-responsivepolymer composition in a solvent to form a temperature-responsivepolymer solution, applying the solution onto the culturing surface of acell culture container, and drying to prepare a coated cell culturecontainer (culture container preparation step I). The culture containerpreparation step may instead be a step of cooling an aqueous solutionincluding a temperature-responsive polymer or a temperature-responsivepolymer composition (temperature-responsive polymer aqueous solution) tothe cloud point of the temperature-responsive polymer or below, castingthe cooled temperature-responsive polymer aqueous solution onto theculturing surface of a cell culture container, and heating to atemperature above the cloud point to prepare a coated cell culturecontainer (culture container preparation step II).

Examples of the solvent in the temperature-responsive polymer solutionin the culture container preparation step I include water; physiologicalsaline; buffer solutions; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 1-butanol, isobutyl alcohol, 2-butanol,t-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol,2,2-dimethyl-1-propanol, 1-pentanol, 2-pentanol, 3-pentanol,2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol,3-methyl-2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol,2-methyl-2-pentanol, allyl alcohol, benzyl alcohol, and salicyl alcohol;ketones such as acetone, ethyl methyl ketone, diethyl ketone, methylpropyl ketone, methyl isobutyl ketone, methyl vinyl ketone,cyclohexanone, 2-methyl cyclopentanone, acetophenone, benzophenone, andisophorone; esters such as methyl acetate, ethyl acetate, propylacetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butylacetate, tert-butyl acetate, vinyl acetate, methyl formate, ethylformate, propyl formate, esters of the aforementioned alcohols andphosphoric acid, and esters of the aforementioned alcohols and carbonicacid; chloroform; benzene; toluene; diethyl ether; and dichloromethane.

Among these, water; alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, 2-butanol, t-butyl alcohol, and allylalcohol; ketones such as acetone, ethyl methyl ketone, diethyl ketone,and methyl vinyl ketone; esters such as methyl acetate, ethyl acetate,isopropyl acetate, tert-butyl acetate, and vinyl acetate; chloroform;benzene; toluene; diethyl ether; and dichloromethane are preferred forfacilitating uniform coating of the culturing surface and for havingexcellent solubility of temperature-responsive polymers. An organicsolvent with a low boiling point (such as at least one selected from thegroup consisting of a low-molecular alcohol with 1 to 4 carbon atoms, alow-molecular ketone with 3 to 5 carbon atoms, and an acetic acid alkylester having an alkyl group with 1 to 4 carbon atoms; in particular, atleast one selected from the group consisting of a low-molecular alcoholwith 1 to 4 carbon atoms, a low-molecular ketone with 3 to 5 carbonatoms, and an acetic acid alkyl ester having an alkyl group with 1 to 4carbon atoms, the low-molecular alcohol, low-molecular ketone, andacetic acid alkyl ester having a boiling point lower than that of water)is more preferable for allowing drying in a short time and facilitatingeven more uniform application on the culturing surface. Methanol andethanol are particularly preferable for their low cost and excellentoperability.

One type of these solvents may be used alone, or a combination of two ormore types may be used.

Since the solvent has excellent solubility with respect to atemperature-responsive polymer, the temperature-responsive polymer tendsnot to become insoluble and precipitate even at a temperature equal toor greater than the cloud point (such as room temperature or 37° C.).This eliminates the need to manage the temperature of thetemperature-responsive polymer during application of thetemperature-responsive polymer, allowing easy preparation of a coatedcell culture container.

In the culture container preparation step I, a hydrophilic molecule ispreferably included in the temperature-responsive polymer to facilitateself-aggregation of cells. Examples of the hydrophilic molecule includenon-ionic, hydrophilic molecules that do not affect the C/A ratio of thetemperature-responsive polymer, such as polyethylene glycol (PEG),dimethyl acrylamide (DMAA), glycerin, Triton X, polypropylene glycol,and the like.

In the culture container preparation step I, the content of thetemperature-responsive polymer in the temperature-responsive polymersolution is preferably 0.01 mass % to 3.0 mass %, more preferably 0.25mass % to 2.5 mass %, relative to the temperature-responsive polymersolution (100 mass %) to facilitate uniform coating of the culturingsurface by the temperature-responsive polymer.

In the culture container preparation step I, the content of thehydrophilic molecule in the temperature-responsive polymer solution ispreferably 0.00001 mass % to 0.00015 mass %, more preferably 0.00003mass % to 0.0001 mass %, relative to the temperature-responsive polymer(100 mass %) to facilitate self-aggregation of cells.

To facilitate uniform coating of the culturing surface by thetemperature-responsive polymer or the temperature-responsive polymercomposition, the temperature-responsive polymer solution in the culturecontainer preparation step I preferably does not include water, and themass ratio of water in the temperature-responsive polymer solution (100mass %) is more preferably 0.5 mass % or less and even more preferably0.1 mass % or less.

The mass ratio of water can be measured by a method known to a personskilled in the art, such as gas chromatography or the Karl Fischermethod.

In the culture container preparation step I, the temperature-responsivepolymer solution may be applied to the entire culturing surface or to aportion of the culturing surface. When the temperature-responsivepolymer solution is applied to a portion of the culturing surface, onecoated culturing surface or a plurality of coated culturing surfaces maybe provided on the culturing surface. A cell culture container with acell non-adhesive culturing surface is preferably used when thetemperature-responsive polymer solution is applied to a portion of theculturing surface.

“Cell non-adhesive” refers to adherent cells (for example, fibroblasts,hepatocytes, vascular endothelial cells, and the like) either notadhering or tending not to adhere under normal culture conditions.

In the culture container preparation step I, preferred conditions fordrying the applied temperature-responsive polymer solution are dryingunder atmospheric pressure at a temperature of 10° C. to 70° C. for 1 to3,000 minutes to uniformly coat the culturing surface with thetemperature-responsive polymer or the temperature-responsive polymercomposition. Quick drying of the applied temperature-responsive polymersolution facilitates uniform coating on the culturing surface with aneven distribution of the temperature-responsive polymer or thetemperature-responsive polymer composition.

The applied temperature-responsive polymer solution may, for example, bedried by letting the cell culture container stand in an incubator at 37°C.

In the culture container preparation step II, examples of the solventfor dissolving the temperature-responsive polymer or thetemperature-responsive polymer composition include water; physiologicalsaline; and buffer solutions such as a phosphate buffer solution,phosphate buffered saline (PBS), and a tris buffer solution.

In the culture container preparation step II, examples of the method ofcooling the temperature-responsive polymer aqueous solution includeplacing the temperature-responsive polymer aqueous solution in arefrigerator at approximately 4° C. and cooling to a temperature at orbelow the cloud point.

In the culture container preparation step II, examples of the method ofcasting the temperature-responsive polymer aqueous solution onto theculturing surface include a method of tilting the culturing surface ofthe cell culture container to spread the temperature-responsive polymeraqueous solution that has a temperature at or below the cloud point anda method of spreading the temperature-responsive polymer aqueoussolution using a spatula.

In the culture container preparation step II, examples of the method ofheating the cast temperature-responsive polymer aqueous solution toabove the cloud point include a method of letting the cell culturecontainer after the casting step stand in an incubator at 37° C.

Examples of the cell culture container include commercially availablemultiwell plates, dishes, flasks, and the like. Examples of the materialof the cell culture container include polystyrene, polyethyleneterephthalate (PET), polypropylene, polybutene, polyethylene,polycarbonate, and glass. Among these, polystyrene and polyethyleneterephthalate (PET) are preferable for being easy to mold precisely, forallowing adoption of various sterilization methods, and for beingsuitable for microscope observation by virtue of being transparent.

Cell adhesion treatment or the like may be applied to the culturingsurface of the cell culture container, or the surface may be untreated.The culturing surface may be coated, processed, or the like to adjustthe cell adhesiveness.

The planar shape of the culturing surface is not restricted and may, forexample, be a substantially rectangular shape or other substantiallypolygonal shape, a substantially circular shape, or the like. Amongthese, a substantially circular shape is preferred to facilitateobtaining a more homogeneous cell structure.

The bottom shape of the culturing surface (the cross-sectional shape ofthe bottom) is not restricted, and examples include a flat bottom, roundbottom, and uneven bottom. Among these, a flat bottom or a slightlycurved concave bottom is preferable to facilitate obtaining a morehomogeneous, spheroidal cell structure.

The area of the culturing surface of the cell culture container ispreferably 32 mm² or less, more preferably 10 mm² or less, and even morepreferably 8 mm² or less, to further facilitate manufacturing of anaggregated cell structure. The lower limit on the area of the culturingsurface of the cell culture container is not restricted, and anycommercially available size may be used.

The area of each coated culturing surface (the area of each portioncoated with a temperature-responsive polymer or a temperature-responsivepolymer composition) is preferably 32 mm² or less, more preferably 10mm² or less, and even more preferably 8 mm² or less, to furtherfacilitate manufacturing of an aggregated cell structure.

In the coated cell culture container, the amount oftemperature-responsive polymer per unit area of the coated culturingsurface is preferably 0.15 ng/mm² to 15.0 ng/mm² and more preferably 1.0ng/mm² to 5.0 ng/mm². Adopting these ranges facilitates achievement ofthe effect of easier formation of an aggregated cell structure.

The zeta potential of the coated culturing surface in the coated cellculture container is preferably 0 mV to 50 mV, more preferably 0 mV to35 mV, and even more preferably 10 mV to 25 mV. A zeta potential of 0 mVor more facilitates adhesion of negatively charged cells. A zetapotential of 50 mV or less can reduce cytotoxicity.

Furthermore, setting the zeta potential in the aforementioned rangesfurther facilitates production of an aggregated (pellet-like) cellstructure by simply culturing cells under appropriate cultureconditions. The reason is that setting the surface zeta potential withinthe aforementioned ranges is inferred to provide the coated culturingsurface with a weak positive charge that does not trigger cytotoxicity,to ensure rapid adhesion of the seeded cells, to improve cell activityand encourage secretion of extracellular matrix, and also toappropriately inhibit cell migration, strengthening the bond betweencells.

The zeta potential refers to the value calculated with the Smoluchowskiequation by measurement using a zeta potential meter (for example, model“ELSZ” by Otsuka Electronics Co.) with a particle (zeta potential: −5 mVto +5 mV) in which polystyrene latex is coated with hydroxypropylcellulose as a reference monitor particle.

The contact angle of water relative to the coated culturing surface ispreferably 50° to 90°, more preferably 60° to 80°, and even morepreferably 62° to 78° to increase the effects of Aspect (VII). Thecontact angle of water relative to the coated culturing surface refersto the average contact angle measured in accordance with JIS R3257 atany number of points on the coated culturing surface.

(Seeding Step)

The seeding step is a step of seeding hepatocytes and fibroblasts in aratio of 10 to 50 fibroblasts per 100 hepatocytes in the coated cellculture container. The cells may all be mixed and seeded at once orseeded a portion at a time. Each type of cell may also be seeded at onceor seeded a portion at a time.

The cells seeded on the coated culturing surface of the coated cellculture container include at least hepatocytes and fibroblasts.

The hepatocytes that are used may, for example, be primary cellscollected from a living organism, hepatocytes derived from iPS cells orES cells, or hepatoma cells such as HepG2 cells or HepRA cells. Amongthese, HepG2 cells and HepRA cells are preferred for having averagehepatocyte functions, such as a transporter function, metabolicactivity, and albumin production, and for ease of obtaining a cellstructure with a homogeneous dispersion of hepatocytes and fibroblasts.

One type of the hepatocytes may be used alone, or a combination of twoor more types may be used.

Examples of the fibroblasts include subcutaneous tissue-derivedfibroblasts, synovium, dental pulp, bone marrow, and subcutaneousadipose-derived mesenchymal stem cells. Among these, subcutaneousadipose-derived mesenchymal stem cells are preferred for havingimmunological tolerance and having little effect on cocultured cells,for being highly adhesive, and for facilitating production of a cellstructure with a more uniform dispersion of hepatocytes and fibroblasts.

One type of fibroblasts may be used alone, or a combination of two ormore types may be used.

In the seeding step, vascular endothelial cells may also be seeded.

One type of vascular endothelial cells may be used alone, or acombination of two or more types may be used.

In the seeding step, adipocytes may also be seeded. Examples of theadipocytes include adipose tissue-derived adherent adipocytes.

One type of adipocytes may be used alone, or a combination of two ormore types may be used.

In the seeding step, immune system cells may also be seeded. The immunesystem cells preferably include at least one selected from the groupconsisting of monocytes, granulocytes, lymphocytes, and macrophages tofacilitate obtaining a cell structure even closer to tissue that hassuffered hepatic failure.

One type of these immune system cells may be used alone, or acombination of two or more types may be used.

In the seeding step, the number of fibroblasts per 100 hepatocytes is 10to 50, preferably 10 to 45. Setting the number of fibroblasts per 100hepatocytes to be 10 or more facilitates curling of the mixed cells toform an aggregated cell structure, and setting the number to be 50 orless allows a cell structure closer to tissue that has suffered hepaticfailure to be obtained.

In the seeding step, the ratio of hepatocytes among all of the seededcells is preferably 50% to 95% of all of the seeded cells (100%) toobtain a cell structure closer to tissue that has suffered hepaticfailure.

In the seeding step, the number of vascular endothelial cells per 100hepatocytes is preferably 10 to 100 to obtain a cell structure closer totissue that has suffered hepatic failure.

In the seeding step, the number of adipocytes per 100 hepatocytes ispreferably 10 to 100, and more preferably 50, to obtain a cell structurecloser to tissue that has suffered hepatic failure.

The number of adipocytes per 100 fibroblasts is preferably 100 to 500 toobtain a cell structure closer to tissue that has suffered hepaticfailure.

Among these ranges, adipocytes are preferably included at a ratio of 50adipocytes per 100 hepatocytes and 100 to 500 adipocytes per 100fibroblasts.

In the seeding step, the number of immune system cells per 100hepatocytes is preferably 10 to 100 to obtain a cell structure closer totissue that has suffered hepatic failure.

In the seeding step, the density of seeded cells on the coated culturingsurface is preferably a confluency of 90% to 100% relative to thesurface area of the coated culturing surface, more preferably aconfluency of 95% to 100%, and even more preferably a confluency of 99%to 100%. Setting the density of seeded cells within the aforementionedranges prevents the cells from each forming colonies, allowing the cellsto aggregate and form a cell structure while remaining homogeneouslydispersed. Furthermore, the seeded mixed cells do not grow easily if thecell density is high, allowing formation of an aggregated cell structurebefore cells grow. Hence, a cell structure including cells in the sameratio as the seeded mixed cells can be formed. The difference in growthrates of cells also has little effect. Since the properties of seededcells may change when the cells grow, impeding cell growth allowsformation of a cell structure including cells with the same propertiesas at the time of seeding.

While the density of the seeded cells on the coated culturing surfacedepends on the type of cell, a density of 20 cells/mm² to 15,000cells/mm² is preferred. For example, when seeding by adding 1.0 mL ofcell fluid to a 24 well cell culture plate with a coated culturingsurface area of 200 mm², the density is preferably 4×10⁴ cells/mL to30×10⁴ cells/mL. Live cells are seeded.

The coated cell culture container may be left to stand in an incubatorat 37° C. and subsequently removed and placed on a clean bench at roomtemperature, for example, with cell seeding then being performed.

Cells are preferably seeded after being diluted in a medium. The mediumfor dilution may be any medium in which cells can be cultured.

(Culturing Step)

The culturing step is a step of culturing the seeded cells to obtain anaggregated cell structure.

It is known that in tissue that has suffered hepatic failure,fibroblasts grow excessively, and additionally many inflammatory immunecells may be present. To reproduce, in a test tube, hepatic failuretissue including immune system cells, it is desirable to produce a cellstructure that includes hepatocytes and fibroblasts and also immunesystem cells, which are suspended cells. However, this is difficult witha known hanging drop method or low-adhesion culture dish, which require1 to 2 weeks to produce cell structures. Apart from different growthrates and different compositions of the optimal medium, the reasons forthis difficulty include a high probability of the mixed macrophagesdifferentiating into fibroblasts during a long culturing period.

In the manufacturing method of Embodiment (VII), immune system cells maybe further added to the coated cell culture container in the culturingstep. The timing at which immune system cells are added is preferablyduring or after the aforementioned seeding step (simultaneous with orafter cell seeding) and before a cell structure is obtained. The timingis more preferably when or after seeded cells adhere to the coatedculturing surface and form a sheet-like cell structure (see (iii) ofFIG. 42) and before the sheet-like cell structure starts to aggregatetowards the central portion of the coated culturing surface and the edgeseparates from the coated culturing surface and starts to warp, forminga cell structure with a warped edge. In other words, the immune systemcells may be seeded during the seeding step or added after the seedingstep.

Specifically, the immune system cells are added simultaneous with orafter cell seeding, preferably within 48 hours after seeding of cells(for example, after the cells that are seeded last).

Adding the immune system cells at the aforementioned timing allows theimmune system cells, which are suspended cells, to precipitate due togravity. When cells adhered to the coated culturing surface aggregatelike a drawstring bag, the immune system cells are incorporated inside,allowing formation of a cell structure that includes immune system cellson the inside. A cell structure even closer to hepatic failure modeltissue can thus be obtained (see FIG. 42).

The conditions for culturing the seeded mixed cells may, for example, bethe use of a typical cell incubator at 37° C. The cells are preferablycultured continuously until forming an aggregated cell structure.Specifically, the cells are preferably cultured for 5 hours to 96 hoursand more preferably for 9 hours to 50 hours.

Surprisingly, even when mixing, seeding, and culturing adherent cells(such as hepatocytes) and suspended cells (such as immune system cells),the manufacturing method of a cell structure of Embodiment (VII) allowsa cell structure to be obtained by seeding and culturing the adherentcells and the suspended cells simultaneously using a medium suitable forthe adherent cells being used or a medium suitable for the suspendedcells being used (preferably a medium suitable for the adherent cells),without the need for preparation of a special medium.

Mixed cells including the hepatocytes and the fibroblasts, which adhereto the coated culturing surface and are cultured, self-aggregate to forman aggregated cell structure. The resulting cell structure includesliving cells inside the aggregated structure.

The cell structure preferably has, for example, an outer diameter of 50μm to 2,500 μm, more preferably 50 μm to 500 μm, and even morepreferably 50 μm to 150 μm, to obtain a cell structure with ahomogeneous distribution of hepatocytes and fibroblasts.

The cell structure obtainable with the manufacturing method of a cellstructure of Embodiment (VII) is a hepatic failure model reproducingtissue that has suffered hepatic failure and can be used for purposesincluding research on hepatic failure and liver regeneration,development of drugs for hepatic failure, and development of treatmentfor hepatic failure.

An example manufacturing method of a cell structure of Embodiment (VII)is described below with reference to FIGS. 41 and 42.

FIG. 41 illustrates an example manufacturing method of a cell structurethat includes hepatocytes and fibroblasts.

A temperature-responsive polymer is applied to a culturing surface of acell culture container to coat the culturing surface, thereby preparinga coated cell culture container having a coated culturing surface (see(i) and (ii) of FIG. 41). Subsequently, mixed cells composed ofhepatocytes and fibroblasts and diluted with medium are added to thecoated culturing surface to seed the mixed cells (see (ii) of FIG. 41).The seeded mixed cells adhere to the entire coated culturing surface(see (iii) of FIG. 41). In this example, the density of the mixed cellsis 100% confluency (see (iii) of FIG. 41). Subsequently, the mixed cellsadhered to the coated culturing surface start to aggregate towards thecentral portion of the coated culturing surface, and the edge separatesfrom the coated culturing surface and starts to warp, yielding a cellstructure with a warped edge (see (iv) of FIG. 41). The cells thencontinue to aggregate to form an aggregated cell structure, which floatsfrom the coated culturing surface (see (v) of FIG. 41).

FIG. 42 illustrates an example manufacturing method of a cell structurethat includes hepatocytes, fibroblasts, and immune system cells(suspended cells) such as macrophages.

A temperature-responsive polymer is applied to a culturing surface of acell culture container to coat the culturing surface, thereby preparinga coated cell culture container having a coated culturing surface (see(i) and (ii) of FIG. 42). Subsequently, mixed cells composed ofhepatocytes and fibroblasts and diluted with medium are added to thecoated culturing surface to seed the mixed cells (see (ii) of FIG. 42).The seeded mixed cells adhere to the entire coated culturing surface toform a sheet-like (single layer) cell structure (see (iii) of FIG. 42).In this example, the density of the mixed cells is 100% confluency (see(iii) of FIG. 42).

After the hepatocytes and fibroblasts form the sheet-like cellstructure, immune system cells such as macrophages are added ((iv) ofFIG. 42). Subsequently, the mixed cells adhered to the coated culturingsurface start to aggregate towards the central portion of the coatedculturing surface, and the edge separates from the coated culturingsurface and starts to warp, yielding a cell structure with a warped edge(see (v) of FIG. 42). At this time, the cell structure incorporates theimmune system cells that are adhered to the sheet-like cell structure orare suspended in the culture dish. The cells then continue to aggregateto form an aggregated cell structure that includes hepatocytes,fibroblasts, and immune system cells. The cell structure then floatsfrom the coated culturing surface (see (vi) of FIG. 42).

EXAMPLES

The present disclosure is described below in greater detail withreference to examples, but the present disclosure is in no way limitedby these examples.

Examples of Aspect (I)

In the following tests, commercially available reagents were usedwithout further purification, unless otherwise noted.

(Test I-A) Production of Temperature-Responsive Polymer

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and5,000 μL of water were added to a 50 mL capacity transparent vial madeof soft glass, and the vial was stirred using a magnetic stirrer. Themixture (liquid) was then purged with Gi-grade, highly pure (purity:99.99995%) nitrogen gas for 10 minutes (flow rate: 2.0 L/min) todeoxygenize the mixture. The DMAEMA that was used included 0.5 weight %of methylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain an intramolecular ioncomplex-type temperature-responsive polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=100,000 (Mw/Mn=10.0).

(Test I-B) Preparation of Coated Culturing Surface

(Test I-B-1) Preparation of Coated Culturing Surface Using a MaskingSheet

A ϕ35 mm low cell-adsorption plate (PrimeSurface® by SUMILON) was usedas a cell culture container.

A sheet (thickness of 1.0 mm) of silicone material (K-125 by TigersPolymer Corporation) modified with a hydrophilic group was prepared, anda cutout that was donut-shaped in plan view (outer diameter (ϕo) of 8mm, inner diameter (ϕi) of 4 mm, and width of 2 mm) was formed in thecentral portion of the sheet. This sheet with a donut-shaped hole wasused as a masking sheet.

The masking sheet was laid on the culturing surface of the plate, 22.5μL of an aqueous solution of the above-described polymer produced inTest I-A (concentration: 3 ng/μL) was added at room temperature, and theaqueous solution was then dried at 45° C. for 3 hours. After drying, thesilicone sheet was peeled off.

(Test I-B-2) Preparation of Coated Culturing Surface Using a CellNon-Adhesive Sheet

A ϕ35 mm low cell-adsorption plate (PrimeSurface® by SUMILON) was usedas a cell culture container.

A sheet of silicone material modified with a hydrophilic group wasprepared, and a cutout that was donut-shaped in plan view (outerdiameter (ϕo) of 8 mm, inner diameter (ϕi) of 4 mm or 3 mm, and width of2 mm or 2.5 mm) was formed in the central portion of the sheet. Thissheet with a donut-shaped hole was used as a pad.

Next, 22.5 μL of an aqueous solution of the aforementioned polymerproduced in Test I-A (concentration: 3 ng/μL) was added at roomtemperature, and the aqueous solution was then dried at 45° C. for 3hours. After drying, the pad was laid on the coated culturing surface.

The surface zeta potential of the coated culturing surface was measuredusing a zeta potential meter (model ELSZ by Otsuka Electronics Co.) anda cell unit for flat plate samples. During the measurement, a quartzcell was used as the cell, a particle (zeta potential: −5 mV to +5 mV)in which polystyrene latex (particle size: approximately 500 nm) wascoated with hydroxypropyl cellulose (Mw=30,000) was used as a referencemonitor particle, and a 10 mM sodium chloride aqueous solution at pH=7and 37° C. was used as a solvent. The zeta potential was calculated withthe Smoluchowski equation.

The resulting zeta potential of the surface of the first coated regioncoated with a temperature-responsive polymer was +20 mV. As is wellknown to a person skilled in the art, the measured value of the zetapotential exhibits a variation of approximately ±10%.

The contact angle of water relative to the first coated region of a cellculture plate was measured as 700±10° using a contact angle meter(DMs-400, by Kyowa Interface Science Co.) in conformity with JIS R3257.

(Test I-C) Seeding and Culturing of Cells

(Test I-C-1) (Reference Example I)

The cell culture container prepared in Test I-B-1 was used.

GFP recombinant rat chondrocyte A (Rat Chondrocyte-A (GFP)) wassuspended in a growth medium (RPMI-1640+10% fetal bovine serum (FBS)+10ng/μL FGF-2; DMEM: by Gibco; FBS: lot number 715929 by BiologicalIndustries; FGF-2: catalog number 400-29 by PeproTech) to produce a cellsuspension.

The cell suspension was added to the plate at room temperature to yielda cell density of at least 1.0×10⁵ cells/cm².

These cells were then cultured in a cell culture incubator at 37° C. and5% CO₂.

Approximately 15 hours after the start of culturing, the chondrocytesstarted to aggregate from the coated culturing surface and formed adonut-shaped (ringed) cell structure by 24 hours after the start ofculturing.

Three hours after the start of culturing, the medium was exchanged witha redifferentiation medium (Dulbecco's Modified Eagle Medium (DMEM)+10%fetal bovine serum (FBS)+10 ng/μL recombinant rat TGF-β1 (Rat TGF-β1recombinant)+50 μg/mL ascorbic acid diphosphate; DMEM: model 11965 byGibco; FBS: lot number 715929 by Bio-Rad; recombinant rat TGF-β1:catalog number 100-21 by PeproTech; ascorbic acid diphosphate: catalognumber 196-01252 by Wako Pure Chemical Industries).

After the medium exchange, the cells were cultured continually foranother 21 hours.

As described above, seeding and culturing was performed once in theabsence of a cell mass in Test I-C-1 (Reference Example I).

FIG. 7 contains photographs taken when using a fluorescence microscopeto observe the state of a cell structure 24 hours (1 day), 2 days, 6days, and 10 days after the start of culturing in Test I-C-1 (ReferenceExample I). In particular, the lower portion of FIG. 7 illustrates apartial enlargement of the photograph of the cell structure after 10days.

It is clear from the photographs in FIG. 7 that the cells constitutingthe resulting cell structure have a cartilage-like circular cell form.

FIG. 8 is a photograph taken after cutting a cross-section of the cellstructure obtained in Test I-C-1 (Reference Example I) along theshort-axis.

It is clear from the photograph in FIG. 8 that the cells constitutingthe resulting cell structure have a unique cartilage cavity structureforming a rounded, pupa-like chamber, that extracellular matrix such aschondroitin and collagen is present in the portion with no surroundingnucleus, and that the cell structure forms cartilage-like tissue.

(Test I-C-2)

The cell culture container prepared in Test I-B-2 was used.

GFP recombinant rat chondrocyte A (Rat Chondrocyte-A (GFP)) wassuspended in a growth medium (RPMI-1640+10% fetal bovine serum (FBS)+10ng/μL FGF-2; DMEM: by Gibco; FBS: lot number 715929 by Bio-Rad; FGF-2:catalog number 400-29 by PeproTech) to produce a cell suspension.

The cell suspension was added to the plate at room temperature to yielda cell density of at least 1.0×10⁵ cells/cm².

These cells were then cultured in a cell culture incubator at 37° C. and5% CO₂.

Fifteen hours after the start of culturing, the chondrocytes started toaggregate from the coated culturing surface and formed a donut-shaped(ringed) cell structure by 24 hours after the start of culturing. Thecell structure precipitated and rested on the widthwise central portionof the bottom of the recess in the pad.

Three to five hours after the start of culturing, the medium wasexchanged with a redifferentiation medium (Dulbecco's Modified EagleMedium (DMEM)+10% fetal bovine serum (FBS)+10 ng/μL recombinant ratTGF-β1 (Rat TGF-β1 recombinant)+50 μg/mL ascorbic acid diphosphate;DMEM: model 11965 by Gibco; FBS: lot number 715929 by Bio-Rad;recombinant rat TGF-β1: catalog number 100-21 by PeproTech; ascorbicacid diphosphate: catalog number 196-01252 by Wako Pure ChemicalIndustries). After the medium exchange, the cells were culturedcontinually for another 24 hours.

After returning the medium to a growth medium, a cell suspension wasadded in the presence of a cell structure at room temperature to yield acell density of at least 1.0×10⁵ cells/cm².

These cells were then cultured in a cell culture incubator at 37° C. and5% CO₂.

Fifteen hours after the start of culturing, the seeded chondrocytesstarted to aggregate from the coated culturing surface, aggregating toenclose the donut-shaped (ringed) cell structure and form a largerdonut-shaped (ringed) cell structure. The cell structure precipitatedand rested on the widthwise central portion of the bottom of the recessin the pad. FIG. 9 illustrates the state of the cell structure 27 hoursafter the start of culturing.

The above seeding and culturing in the presence of a cell structure wasrepeated 3 times. FIG. 9 illustrates the state of the cell structureafter 1, 2, and 3 repetitions.

FIG. 9 contains photographs taken when using a fluorescence microscopeto observe the state of a cell structure 27 hours, 44 hours, 70 hours,and 122 hours after the start of culturing in Test I-C-2. The upperportion illustrates the state of the cell structure when using a padwith a 2 mm wide donut-shaped cutout, and the lower portion illustratesthe state of the cell structure when using a pad with a 2.5 mm widedonut-shaped cutout.

It is clear from the photographs in FIG. 9 that the size of thedonut-shaped (ringed) chondrocyte mass was greater after seeding andculturing of new cells.

(Test I-C-3)

The cell culture container prepared in Test I-B-2 was used.

Adipose-derived mesenchymal stem cells (adipose-derived vascular stromalcells (ADSC)) of a GFP recombinant Lewis rat were suspended in a growthmedium (Dulbecco's Modified Eagle Medium (DMEM)+10% fetal bovine serum(FBS); DMEM: model 11965 by Gibco; FBS: lot number 715929 by Bio-Rad) toproduce a cell suspension.

The cell suspension was added to the plate at room temperature to yielda cell density of at least 1.0×10⁵ cells/cm².

These cells were then cultured in a cell culture incubator at 37° C. and5% CO₂.

Six hours after the start of culturing, the chondrocytes started toaggregate from the coated culturing surface and formed a donut-shaped(ringed) cell structure by 8 hours after the start of culturing. Thecell structure precipitated and rested on the widthwise central portionof the bottom of the recess in the pad.

One and a half hours after the start of culturing, the medium wasexchanged with a cartilage differentiation medium (Dulbecco's ModifiedEagle Medium (DMEM)+10% fetal bovine serum (FBS)+1% ITS Premix+50 μg/mLascorbic acid diphosphate+10 ng/μL recombinant rat TGF-β1 (Rat TGF-β1recombinant)+10 M dexamethasone; DMEM: model 11965 by Gibco; FBS: lotnumber 715929 by Bio-Rad; ITS Premix: catalog number 354341 by BDBiosciences; ascorbic acid diphosphate: catalog number 196-01252 by WakoPure Chemical Industries; recombinant rat TGF-β1: catalog number 100-21by PeproTech; dexamethasone: catalog number 047-18863 by Wako PureChemical Industries).

After the medium exchange, the cells were cultured continually foranother 24 hours.

After returning the medium to a growth medium, a cell suspension wasadded in the presence of a cell structure at room temperature to yield acell density of at least 1.0×10⁵ cells/cm²

These cells were then cultured in a cell culture incubator at 37° C. and5% CO₂.

Six hours after the start of culturing, the seeded chondrocytes startedto aggregate from the coated culturing surface, aggregating to enclosethe donut-shaped (ringed) cell structure and form a larger donut-shaped(ringed) cell structure. FIG. 10 illustrates the state of the cellstructure 8 hours after the start of culturing.

The above seeding and culturing in the presence of a cell structure wasrepeated 3 times. FIG. 10 illustrates the state of the cell structureafter 1, 2, and 3 repetitions.

FIG. 10 contains photographs taken when using a fluorescence microscopeto observe the state of a cell structure 8 hours, 20 hours, 32 hours,and 42 hours after the start of culturing in Test I-C-3. The upperportion illustrates the state of the cell structure when using a padwith a 2 mm wide donut-shaped cutout, and the lower portion illustratesthe state of the cell structure when using a pad with a 2.5 mm widedonut-shaped cutout. The lowermost portion is a photograph taken whenusing a stereomicroscope to observe the state of the cell structureafter 42 hours.

It is clear from the photographs in FIG. 10 that the size of thedonut-shaped (ringed) chondrocyte mass was greater after seeding andculturing of new cells.

In particular, it is clear that after repeating the seeding andculturing 3 times (42 hours after the start of culturing), thedonut-shaped (ringed) chondrocyte mass protruded outside of the recessin the pad.

A graft material (not illustrated) was successfully produced byperforming the seeding and culturing step one more time using ADSCcells, which are mesenchymal cells, on the donut-shaped (ringed)chondrocyte mass obtained in Test I-C-2 and Test I-C-3.

(Test I-D) Production of Composite Material

As a tubular structure, a biotube (outer diameter: 3 mm; inner diameter:2 mm; length: 20 mm) having collagen as the principal component (see,for example, the method disclosed in the Examples of JP2004261260A) wasprepared. A silicone rod-like structure (cylindrical; length: 20 mm;outer diameter: 1.8 mm) was prepared as a core material (see (i) of FIG.6).

Next, the rod-like structure was inserted into the hollow portion of thebiotube from one end to the other (see (ii) of FIG. 6).

Four of the donut-shaped chondrocyte masses produced in Test I-C-2 werefitted onto the biotube with a gap of approximately 1 mm between eachchondrocyte mass to produce a composite body (see (iii) of FIG. 6).

Three minutes after completion of fitting, culturing of the compositebody in a cell culture incubator at 37° C. and 5% CO₂ was started.Thereafter, the composite body was cultured for 21 days. By 21 daysafter the start of culturing, the biotube and the chondrocyte mass wereintegrated to produce a composite material (see (iv) of FIG. 6).

FIG. 11A is a photograph taken when observing the state of the compositematerial with the naked eye 6 days after the start of culturing in TestI-D.

FIG. 11B is a photograph taken when observing the state of the compositematerial with the naked eye 21 days after the start of culturing in TestI-D.

FIG. 12 is an enlargement of a photograph taken when observing the stateof a portion of the composite material with the naked eye 21 days afterthe start of culturing in Test I-D.

As illustrated in FIG. 12, cartilage tissue was formed on the outersurface of the biotube.

The interval between chondrocyte masses that was approximately 1 mm whenthe composite body was produced was approximately 1 mm.

FIGS. 13A to 13D are photographs taken when observing, with the nakedeye, the state of the composite material produced in Test I-D whenmanipulating the composite material with tweezers. FIG. 13A illustratesthe outer peripheral surface without manipulation, FIG. 13B illustratesthe luminal surface without manipulation, FIG. 13C illustrates the statewhen the entire material is crushed, and FIG. 13D illustrates the statewhen pulling towards a portion of the side surface.

It is clear from FIGS. 13A to 13D that the composite material hadsufficient mechanical strength to withstand the aforementioned normaloperations, and that the cartilage tissue also remained firmly adheredto the biotube.

FIGS. 14A to 14D are photographs taken when using a microscope toobserve the state of a composite material produced in Test I-D whensubjecting the composite material to a hematoxylin and eosin stain (H&Estain). FIG. 14A is an exterior photograph of the composite material,FIG. 14B is a cross-sectional view of the composite material in a planealong the A-A line in FIG. 14A, and FIGS. 14C and 14D are partialenlargements of the photograph in FIG. 14B.

The portions stained pink in FIGS. 14C and 14D (indicated by solidarrows in the figures) are collagen, and the portions stained violet arecells. It is clear from these figures that the collagen fibers of thebiotube and the extracellular matrix of the chondrocyte are integrated.

Examples of Aspect (II)

Aspect (II) is described below in greater detail with reference toexamples, but the present disclosure is in no way limited by theseexamples.

(Production of Temperature-Responsive Polymer)

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and 5 mLof water were added to a 50 mL capacity transparent vial made of softglass, and the vial was stirred using a magnetic stirrer. The mixture(liquid) was then purged with Gi-grade, highly pure (purity: 99.99995%)nitrogen gas for 10 minutes (flow rate: 2.0 L/min) to deoxygenize themixture. The DMAEMA that was used included 0.5 mass % ofmethylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain atemperature-responsive (homo)polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=1.0×10⁵ g/mol (Mw/Mn=10.0).

The nuclear magnetic resonance (NMR) spectrum of the above-describedtemperature-responsive polymer was measured using a nuclear magneticresonance apparatus (model Gemini-300 by Varian) with heavy water (D₂O)as a standard substance. A representative peak is indicated below.

¹H-NMR (in D₂O) δ 0.8-1.2 (br, —CH₂—C(CH₃)—), 1.6-2.0 (br,—CH₂—C(CH₃)—), 2.2-2.4 (br, —N(CH₃)₂), 2.5-2.7 (br, —CH₂—N(CH₃)₂),4.0-4.2 (br, —O—CH₂—).

Here, from the number of protons A in the methyl group (δ 0.8-1.2) ofthe main chain (3 per monomer molecule in the case of a DMAEMAhomopolymer) and the number of methyl protons B in the dimethylaminogroup (δ 2.2-2.4) of the side chain (6 per monomer molecule in the caseof a DMAEMA homopolymer), the ratio was calculated between the number offunctional groups that are amino groups in the side chain and the numberof functional groups that are carboxyl groups in the side chain producedby a hydrolysis reaction, which proceeds simultaneously with thepolymerization reaction, of an ester bond of the side chain.

The resulting ratio was 94:6 in the case of the above-describedtemperature-responsive polymer. Converting into the C/A ratio for an ioncomplex in a two-component mixed system that includes a cationic polymerand an anionic polymer yields a C/A ratio of 15.6.

The cloud point of the above-described temperature-responsive polymerwas measured with the following method.

A 3% aqueous solution of the temperature-responsive polymer wasproduced, and the absorbance of the aqueous solution at 660 nm wasmeasured between 20° C. and 40° C.

Between 20° C. and 30° C., the aqueous solution was transparent, with anabsorbance of nearly 0. Starting around 31° C., however, the aqueoussolution became cloudy, and the absorbance increased suddenly at 32° C.The temperature-responsive polymer was thus confirmed to have a cloudpoint of approximately 32° C.

Once the temperature-responsive polymer was increased in temperature to37° C., the polymer aqueous solution was suspended with goodresponsiveness. Subsequently, the entire aqueous solution hardened. Whenmaintained at room temperature (25° C.), the hardened product retainedits hard state for several tens of hours. Subsequently, the hardenedproduct gradually dissolved, changing into a homogeneous aqueoussolution. Upon being cooled to 4° C., the hardened polymer rapidlydissolved. Repeating the aforementioned operation to raise and lower thetemperature caused no change in responsiveness, thereby confirming thatthe polymer reversibly underwent phase transitions.

(Manufacturing of Coated Cell Culture Container)

The above-described temperature-responsive polymers were dissolved inpure water to produce temperature-responsive polymer solutions. Theconcentration of the temperature-responsive polymer in eachtemperature-responsive polymer solution was adjusted as follows:temperature-responsive polymer a, 0.125 ng/μL; temperature-responsivepolymer b, 0.25 ng/μL; temperature-responsive polymer c, 0.5 ng/μL;temperature-responsive polymer d, 1.0 ng/μL.

Next, 25 μL of the temperature-responsive polymers a to d was added tothe wells of 384-well plates (PrimeSurface®, MS-9384U, by SumitomoBakelite Co.).

The applied temperature-responsive polymer aqueous solution was thendried by leaving the plates in an incubator (40° C.) for 6 hours toprepare coated cell culture containers a to d having the coated regionA.

The above-described temperature-responsive polymers were dissolved inphysiological saline to produce temperature-responsive polymersolutions. The concentration of the temperature-responsive polymer ineach temperature-responsive polymer solution was adjusted as follows:temperature-responsive polymer e, 6 ng/μL; temperature-responsivepolymer f, 12 ng/μL; temperature-responsive polymer g, 24 ng/μL.

Next, 25 μL of the temperature-responsive polymers e to g was added tothe wells of 384-well plates (PrimeSurface®, MS-9384U, by SumitomoBakelite Co.). The wells of the plates had a rounded bottom with aradius of curvature (average) R of approximately 1.6 mm and had amaximum width L of approximately 2.5 mm.

The plates were left in an incubator (37° C.) for 3 hours to preparecoated cell culture containers e to g having the coated region A. In thecoated cell culture containers e to g, the temperature-responsivepolymer solution added to the wells was not dried.

The above-described temperature-responsive polymers were dissolved inpure water to produce temperature-responsive polymer solutions. Theconcentration of the temperature-responsive polymer in eachtemperature-responsive polymer solution was adjusted as follows:temperature-responsive polymer h, 0.9 pg/μL; temperature-responsivepolymer i, 1.8 pg/μL; temperature-responsive polymer j, 7.5 pg/μL;temperature-responsive polymer k, 15 pg/μL; temperature-responsivepolymer 1, 31 pg/μL; temperature-responsive polymer m, 67 pg/μL;temperature-responsive polymer n, 125 pg/μL; and temperature-responsivepolymer o, 500 pg/μL.

Next, 2.5 μL of the temperature-responsive polymers h to o was added tothe wells of 384-well plates (PrimeSurface®, MS-9384U, by SumitomoBakelite Co.).

The applied temperature-responsive polymer aqueous solution was thendried by leaving the plates in an incubator (40° C.) for 6 hours toprepare coated cell culture containers h to o having the coated regionA.

Next, 25 μL of physiological saline was added to the wells of a 384-wellplate (PrimeSurface®, MS-9384U, by Sumitomo Bakelite Co.).

The plate was then left for 3 hours in an incubator (37° C.) to preparea cell culture container p. The cell culture container p was a cellculture container not coated by a temperature-responsive polymer.

[Culture Method of Epithelial Cells]

Examples II-1 to II-35

Human hepatoma cells (number “HepG2-500” by Cosmo Bio) were mixed into amedium (DMAEM+10% FBS (lot number 715929 by Gibco)) to produce cellsolutions at the following concentrations: 0.5×10³ cells/50 μL (cellsolution I), 1.0×10³ cells/50 μL (cell solution II), 2.0×10³ cells/50 μL(cell solution III), 4.0×10³ cells/50 μL (cell solution IV), and 8.0×10³cells/50 μL (cell solution V).

Next, 50 μL of the cell solutions I to V was added to the wells of thecoated cell culture containers a to g to seed cells. Table 1 lists thecombination of coated cell culture container and cell solution in eachExample.

Subsequently, cells were cultured for 24 hours in a cell incubator (37°C., 5% CO₂). The cells were then observed with a microscope (ECLIPSE-Tiby Nikon Corporation).

Examples II-36 to II-75

Human hepatoma cells (number “HepG2-500” by Cosmo Bio) were mixed into amedium (DMAEM+10% FBS (lot number 715929 by Gibco)) to produce cellsolutions at the following concentrations: 0.5×10³ cells/25 μL (cellsolution VI), 1.0×10³ cells/25 μL (cell solution VII), 2.0×10³ cells/25μL (cell solution VIII), 4.0×10³ cells/25 μL (cell solution IX), and8.0×10³ cells/25 μL (cell solution X).

Next, 25 μL of the cell solutions VI to X was added to the wells of thecoated cell culture containers h to o to seed cells. Table 1 lists thecombination of coated cell culture container and cell solution in eachExample.

Subsequently, cells were cultured for 24 hours in a cell incubator (37°C., 5% CO₂). The cells were then observed with a microscope (ECLIPSE-Tiby Nikon Corporation).

Comparative Examples II-1 to II-5

Fifty μL of the cell solutions I to V was added to the wells of thecoated cell culture container p to seed cells. Table 1 lists thecombination of coated cell culture container and cell solution in eachComparative Example. Subsequently, cells were cultured for 24 hours in acell incubator (37° C., 5% CO₂). The cells were then observed with amicroscope (ECLIPSE-Ti by Nikon Corporation).

[Manufacturing Method of a Cell Structure]

Examples II-76 to II-80

Fifty μL of the cell solutions I to V was added to the wells of thecoated cell culture container e to seed cells. Table 2 lists thecombination of coated cell culture container and cell solution in eachExample.

Subsequently, cells were cultured for 24 hours in a cell incubator (37°C., 5% CO₂) and observed with a microscope (ECLIPSE-Ti by NikonCorporation). It was confirmed that an aggregated cell structure wasformed at the bottom of all of the wells. The below-described evaluationof peeling resistance of the cell structure was also performed. Table 2lists the results.

Examples II-81 to II-100

Twenty-five μL of the cell solutions VI to X was added to the wells ofthe coated cell culture containers h to k to seed cells. Table 2 liststhe combination of coated cell culture container and cell solution ineach Example.

Subsequently, cells were cultured for 24 hours in a cell incubator (37°C., 5% CO₂) and observed with a microscope (ECLIPSE-Ti by NikonCorporation). It was confirmed that an aggregated cell structure wasformed at the bottom of all of the wells. The below-described evaluationof peeling resistance of the cell structure was also performed. Table 2lists the results.

Comparative Examples II-6 to II-10

Fifty μL of the cell solutions I to V was added to the wells of thecoated cell culture container p to seed cells. Table 2 lists thecombination of cell culture container and cell solution in eachComparative Example.

Subsequently, cells were cultured for 24 hours in a cell incubator (37°C., 5% CO₂) and observed with a microscope (ECLIPSE-Ti by NikonCorporation).

HepG2 aggregated at the bottom without adhering to the culturingsurface. The aggregated cell structure was not adhered to the bottom,and the cell structure moved when the plate was simply shaken. In somecases, the cell structure collapsed when pipetting was performed,perhaps because of the low secretion of extracellular matrix.

[Evaluation]

(Adhesion of Cells to Coated Region)

The HepG2 cells cultured in Examples II-1 to II-75 and ComparativeExamples II-1 to II-5 were observed with a microscope (ECLIPSE-Ti byNikon Corporation). The case of the HepG2 cells adhering to the coatedregion and being culturable was evaluated as good (G), and the case ofHepG2 not adhering to the coated region was evaluated as poor (P).

(Appearance of Cell Structure)

The cell structures obtained in Examples II-76 to II-100 and ComparativeExamples II-6 to II-10 were observed with a microscope (ECLIPSE-Ti byNikon Corporation). Subsequently, pipetting was performed forcefullyusing a pipettor (“Reference” by Eppendorf), and the cell structureswere observed again. The appearance of the cell structures was thenevaluated with the following criteria.

G (good): an aggregated cell structure was formed, and the cellstructure did not collapse even when subjected to forceful pipetting.P (poor): an aggregated cell structure was formed but collapsed whensubjected to forceful pipetting.

(Peeling Resistance of Cell Structure)

The cell structures obtained in Examples II-76 to II-100 and ComparativeExamples II-6 to II-10 were repeatedly subjected to manual pipettingusing a pipettor (“Reference” by Eppendorf), and the number of timesuntil the cell structures peeled off from the culturing surface wascounted.

E (excellent): the cell structure did not peel off even when subjectedto pipetting more than 10 times.G (good): the cell structure peeled off when subjected to pipetting 2 to10 times.F (fair): the cell structure peeled off when subjected to pipettingonce.P (poor): the cell structure was not adhered to the culturing surface.

TABLE 1 Coated Cell Culture Container Temperature- Cell SolutionResponsive Number Polymer Added of Cells Evaluation Concentration Amountper Adhesion Type (pg/mm²) Type (μL) Well of Cells Example II-1 a 372.0I 50 0.5 × 10³ G Example II-2 a 372.0 II 50 1.0 × 10³ G Example II-3 a372.0 III 50 2.0 × 10³ G Example II-4 a 372.0 IV 50 4.0 × 10³ G ExampleII-5 a 372.0 V 50 8.0 × 10³ G Example II-6 b 744.0 I 50 0.5 × 10³ GExample II-7 b 744.0 II 50 1.0 × 10³ G Example II-8 b 744.0 III 50 2.0 ×10³ G Example II-9 b 744.0 IV 50 4.0 × 10³ G Example II-10 b 744.0 V 508.0 × 10³ G Example II-11 c 1488.1 I 50 0.5 × 10³ G Example II-12 c1488.1 II 50 1.0 × 10³ G Example II-13 c 1488.1 III 50 2.0 × 10³ GExample II-14 c 1488.1 IV 50 4.0 × 10³ G Example II-15 c 1488.1 V 50 8.0× 10³ G Example II-16 d 2976.2 I 50 0.5 × 10³ G Example II-17 d 2976.2II 50 1.0 × 10³ G Example II-18 d 2976.2 III 50 2.0 × 10³ G ExampleII-19 d 2976.2 IV 50 4.0 × 10³ G Example II-20 d 2976.2 V 50 8.0 × 10³ GExample II-21 e 17857.1 I 50 0.5 × 10³ G Example II-22 e 17857.1 II 501.0 × 10³ G Example II-23 e 17857.1 III 50 2.0 × 10³ G Example II-24 e17857.1 IV 50 4.0 × 10³ G Example II-25 e 17857.1 V 50 8.0 × 10³ GExample II-26 f 35714.3 I 50 0.5 × 10³ G Example II-27 f 35714.3 II 501.0 × 10³ G Example II-28 f 35714.3 III 50 2.0 × 10³ G Example II-29 f35714.3 IV 50 4.0 × 10³ G Example II-30 f 35714.3 V 50 8.0 × 10³ GExample II-31 g 71428.6 I 50 0.5 × 10³ G Example II-32 g 71428.6 II 501.0 × 10³ G Example II-33 g 71428.6 III 50 2.0 × 10³ G Example II-34 g71428.6 IV 50 4.0 × 10³ G Example II-35 g 71428.6 V 50 8.0 × 10³ GExample II-36 h 0.27 VI 25 0.5 × 10³ G Example II-37 h 0.27 VII 25 1.0 ×10³ G Example II-38 h 0.27 VIII 25 2.0 × 10³ G Example II-39 h 0.27 IX25 4.0 × 10³ G Example II-40 h 0.27 X 25 8.0 × 10³ G Example II-41 i0.54 VI 25 0.5 × 10³ G Example II-42 i 0.54 VII 25 1.0 × 10³ G ExampleII-43 i 0.54 VIII 25 2.0 × 10³ G Example II-44 i 0.54 IX 25 4.0 × 10³ GExample II-45 i 0.54 X 25 8.0 × 10³ G Example II-46 j 2.2 VI 25 0.5 ×10³ G Example II-47 j 2.2 VII 25 1.0 × 10³ G Example II-48 j 2.2 VIII 252.0 × 10³ G Example II-49 j 2.2 IX 25 4.0 × 10³ G Example II-50 j 2.2 X25 8.0 × 10³ G Example II-51 k 4.5 VI 25 0.5 × 10³ G Example II-52 k 4.5VII 25 1.0 × 10³ G Example II-53 k 4.5 VIII 25 2.0 × 10³ G Example II-54k 4.5 IX 25 4.0 × 10³ G Example II-55 k 4.5 X 25 8.0 × 10³ G ExampleII-56 l 9.2 VI 25 0.5 × 10³ G Example II-57 l 9.2 VII 25 1.0 × 10³ GExample II-58 l 9.2 VIII 25 2.0 × 10³ G Example II-59 l 9.2 IX 25 4.0 ×10³ G Example II-60 l 9.2 X 25 8.0 × 10³ G Example II-61 m 19.9 VI 250.5 × 10³ G Example II-62 m 19.9 VII 25 1.0 × 10³ G Example II-63 m 19.9VIII 25 2.0 × 10³ G Example II-64 m 19.9 IX 25 4.0 × 10³ G Example II-65m 19.9 X 25 8.0 × 10³ G Example II-66 n 37.2 VI 25 0.5 × 10³ G ExampleII-67 n 37.2 VII 25 1.0 × 10³ G Example II-68 n 37.2 VIII 25 2.0 × 10³ GExample II-69 n 37.2 IX 25 4.0 × 10³ G Example II-70 n 37.2 X 25 8.0 ×10³ G Example II-71 o 148.8 VI 25 0.5 × 10³ G Example II-72 o 148.8 VII25 1.0 × 10³ G Example II-73 o 148.8 VIII 25 2.0 × 10³ G Example II-74 o148.8 IX 25 4.0 × 10³ G Example II-75 o 148.8 X 25 8.0 × 10³ GComparative p 0 I 50 0.5 × 10³ P Example II-1 Comparative p 0 II 50 1.0× 10³ P Example II-2 Comparative p 0 III 50 2.0 × 10³ P Example II-3Comparative p 0 IV 50 4.0 × 10³ P Example II-4 Comparative p 0 V 50 8.0× 10³ P Example II-5

TABLE 2 Coated Cell Culture Container Temperature- Cell SolutionEvaluation Responsive Number Peeling Polymer Added of Cells AppearanceResistance Concentration Amount per of Cell of Cell Type (pg/mm²) Type(μL) Well Structure Structure Example II-76 e 17857.1 I 50 0.5 × 10³ G FExample II-77 e 17857.1 II 50 1.0 × 10³ G F Example II-78 e 17857.1 III50 2.0 × 10³ G F Example II-79 e 17857.1 IV 50 4.0 × 10³ G F ExampleII-80 e 17857.1 V 50 8.0 × 10³ G F Example II-81 h 0.27 VI 25 0.5 × 10³G F Example II-82 h 0.27 VII 25 1.0 × 10³ G F Example II-83 h 0.27 VIII25 2.0 × 10³ G F Example II-84 h 0.27 IX 25 4.0 × 10³ G F Example II-85h 0.27 X 25 8.0 × 10³ G F Example II-86 i 0.54 VI 25 0.5 × 10³ G FExample II-87 i 0.54 VII 25 1.0 × 10³ G F Example II-88 i 0.54 VIII 252.0 × 10³ G F Example II-89 i 0.54 IX 25 4.0 × 10³ G F Example II-90 i0.54 X 25 8.0 × 10³ G F Example II-91 j 2.2 VI 25 0.5 × 10³ G G ExampleII-92 j 2.2 VII 25 1.0 × 10³ G G Example II-93 j 2.2 VIII 25 2.0 × 10³ GG Example II-94 j 2.2 IX 25 4.0 × 10³ G G Example II-95 j 2.2 X 25 8.0 ×10³ G G Example II-96 k 4.5 VI 25 0.5 × 10³ G E Example II-97 k 4.5 VII25 1.0 × 10³ G E Example II-98 k 4.5 VIII 25 2.0 × 10³ G E Example II-99k 4.5 IX 25 4.0 × 10³ G E Example II-100 k 4.5 X 25 8.0 × 10³ G EComparative p 0 I 50 0.5 × 10³ P P Example II-6 Comparative p 0 II 501.0 × 10³ P P Example II-7 Comparative p 0 III 50 2.0 × 10³ p p ExampleII-8 Comparative p 0 IV 50 4.0 × 10³ P P Example II-9 Comparative p 0 V50 8.0 × 10³ P P Example II-10

[Epithelial-Mesenchymal Transition Inducer]

Example II-101

Human hepatoma cells (number “HepG2-500” by Cosmo Bio) were mixed into amedium (DMAEM+10% FBS (lot number 715929 by Gibco)) to produce a cellsolution at a concentration of 4.0×10³ cells/25 μL.

A 0.5 μL droplet of the above-described temperature-responsive polymer d(1.0 ng/μL) was spotted onto the central portion of the wells of a24-well plate (“Sumilon cell tight”, MS-9024X, by Sumitomo Bakelite Co.)using a micropipettor (Digifit by Sibata Scientific Technology).

The applied temperature-responsive polymer aqueous solution was thendried by leaving the plate in an incubator (37° C.) for 3 hours toprepare a coated cell culture container q having the coated region A.

Next, 1,000 μL of the cell solution I was added to the wells of thecoated cell culture container q to seed cells.

Subsequently, cells were cultured for 96 hours in a cell incubator (37°C., 5% CO₂). The cells were then observed with a microscope (ECLIPSE-Tiby Nikon Corporation).

(Transition from Epithelial Cells to Stromal Cells)

The HepG2 cells cultured in Example II-101 were observed with amicroscope (ECLIPSE-Ti by Nikon Corporation). The adhesion state of theHepG2 cells adhered to the coated region was evaluated to judge whethertransition from epithelial cells to stromal cells had occurred.

TABLE 3 Coated Cell Culture Container Temperature- Cell SolutionResponsive Number Evaluation Polymer Added of Cells Transition fromConcentration Amount per Epithelial Cells to Type (pg/mm²) Type (μL)Well Stromal Cells FIG. Example q 2.8 I 1000 1.0 × 10⁴ cells in coatedregion: FIG. II-101 confluent 17 cells in non-coated region: scattered(isolated) like cobblestones

FIG. 17 is a photograph illustrating the state when culturing epithelialcells for 96 hours on a temperature-responsive polymer or atemperature-responsive polymer composition used in the presentdisclosure. In FIG. 17, the portion surrounded by the dashed lineindicates a coated region A, the arrow with a black outline indicatescells adhered to and growing in the coated region A, and the solid blackarrows indicate cells adhered to and growing in the non-coated region.

It is clear from FIG. 17 that after culturing for 96 hours, the cellscultured in the coated region A were confluent and had a fibroblast-likeform, whereas the cells cultured in the non-coated region were scattered(isolated) like cobblestones and maintained the characteristics ofepithelial cells. These results indicate that the temperature-responsivepolymer or the temperature-responsive polymer composition used in thepresent disclosure has the effect of epithelial-stromal transition.

Examples of Aspect (III)

Aspect (III) is described below in greater detail with reference toexamples, but the present disclosure is in no way limited by theseexamples.

Example III-1 (Manufacturing of Production Apparatus ofThree-Dimensional Tissue Body)

A stainless-steel tube (by Fuji Filter Manufacturing Co., 3ϕ) with ameshed surface was used as a shaft, and “PrimeSurface®” (numberMS-9024X, by Sumitomo Bakelite Co.) was used as a culturing surface, aportion of the culturing surface being cut out to form a through hole(approximately 3 mm diameter), to manufacture an apparatus in which theculturing surface and the shaft are in contact. The culturing surfacewas substantially circular, with an outer diameter of approximately 25mm. The apparatus was shaped as in FIG. 18.

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and 5 mLof water were added to a 50 mL capacity transparent vial made of softglass, and the vial was stirred using a magnetic stirrer. The mixture(liquid) was then purged with Gi-grade, highly pure (purity: 99.99995%)nitrogen gas for 10 minutes (flow rate: 2.0 L/min) to deoxygenize themixture. The DMAEMA that was used included 0.5 mass % ofmethylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain atemperature-responsive (homo)polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=1.0×10⁵ g/mol (Mw/Mn=10.0).

The nuclear magnetic resonance (NMR) spectrum of the above-describedtemperature-responsive polymer was measured using a nuclear magneticresonance apparatus (model Gemini-300 by Varian) with heavy water (D₂O)as a standard substance. A representative peak is indicated below.

¹H-NMR (in D₂O) δ 0.8-1.2 (br, —CH₂—C(CH₃)—), 1.6-2.0 (br,—CH₂—C(CH₃)—), 2.2-2.4 (br, —N(CH₃)₂), 2.5-2.7 (br, —CH₂—N(CH₃)₂),4.0-4.2 (br, —O—CH₂—).

Here, from the number of protons A in the methyl group (δ 0.8-1.2) ofthe main chain (3 per monomer molecule in the case of a DMAEMAhomopolymer) and the number of methyl protons B in the dimethylaminogroup (δ 2.2-2.4) of the side chain (6 per monomer molecule in the caseof a DMAEMA homopolymer), the ratio was calculated between the number offunctional groups that are amino groups in the side chain and the numberof functional groups that are carboxyl groups in the side chain producedby a hydrolysis reaction, which proceeds simultaneously with thepolymerization reaction, of an ester bond of the side chain.

The resulting ratio was 94:6 in the case of the above-describedtemperature-responsive polymer. Converting into the C/A ratio for an ioncomplex in a two-component mixed system that includes a cationic polymerand an anionic polymer yields a C/A ratio of 15.6.

The cloud point of the above-described temperature-responsive polymerwas measured with the following method.

A 3% aqueous solution of the temperature-responsive polymer wasproduced, and the absorbance of the aqueous solution at 660 nm wasmeasured between 20° C. and 40° C.

Between 20° C. and 30° C., the aqueous solution was transparent, with anabsorbance of nearly 0. Starting around 31° C., however, the aqueoussolution became cloudy, and the absorbance increased suddenly at 32° C.The temperature-responsive polymer was thus confirmed to have a cloudpoint of approximately 32° C.

Once the temperature-responsive polymer was increased in temperature to37° C., the polymer aqueous solution was suspended with goodresponsiveness. Subsequently, the entire aqueous solution hardened. Whenmaintained at room temperature (25° C.), the hardened product retainedits hard state for several tens of hours. Subsequently, the hardenedproduct gradually dissolved, changing into a homogeneous aqueoussolution. Upon being cooled to 4° C., the hardened polymer rapidlydissolved. Repeating the aforementioned operation to raise and lower thetemperature caused no change in responsiveness, thereby confirming thatthe polymer reversibly underwent phase transitions.

The above-described temperature-responsive polymer was dissolved in purewater to produce a temperature-responsive polymer solution (finalconcentration 15 ng/μL). This solution was applied to the entireculturing surface of the above-described apparatus, and the appliedtemperature-responsive polymer aqueous solution was then dried byleaving the apparatus in an incubator (40° C.) for 1 hour to prepare aproduction apparatus, of a three-dimensional tissue body, having acoated culturing surface.

Example III-2

A stainless-steel tube (by Fuji Filter Manufacturing Co., 3ϕ) with ameshed surface was used as a shaft, and “PrimeSurface®” (numberMS-9024X, by Sumitomo Bakelite Co.) was used as a culturing surface, aportion of the culturing surface being cut out to form a through hole(approximately 3.2 mm diameter), to manufacture an apparatus with a gapbetween the culturing surface and the shaft. The culturing surface wassubstantially circular, with an outer diameter of approximately 25 mm.The gap between the culturing surface and the shaft was 0.2 mm. Theapparatus was shaped as in FIG. 20.

As in Example III-1, the culturing surface was coated with atemperature-responsive polymer to prepare a production apparatus, of athree-dimensional tissue body, having a coated culturing surface.

Example III-3 (Production of Ringed Three-Dimensional Tissue Body)

The production apparatus of a three-dimensional tissue body manufacturedin Example III-1 was placed in a conical tube (product code “2345-050”by Iwaki & Co) with an inner diameter of 30 mm and immersed in a medium(DMEM+10% FBS (lot number 715929 by Biological Industries)+50 μg/mL ofascorbic acid diphosphate (catalog number 196-1252 by Wako Pure ChemicalIndustries)). Subsequently, adipose-derived mesenchymal stem cells(ADSC) of a GFP recombinant Lewis rat were mixed into a medium, similarto the medium in which the production apparatus was immersed, to producea cell suspension. Next, 2 mL of the cell suspension at 60×10⁵ cells/mLwas added to seed cells.

The cells were then cultured for 24 hours in a cell incubator (37° C.,5% CO₂), and obtainment of a ringed three-dimensional tissue body woundaround the shaft was confirmed.

The resulting ringed three-dimensional tissue body was nearly identicalto the one in FIG. 24. The resulting ringed three-dimensional tissuebody had ADSC as the principal component.

Example III-4 (Production of Ringed Three-Dimensional Tissue Body)

Except for using the production apparatus of a three-dimensional tissuebody produced in Example III-2, a ringed three-dimensional tissue bodywas produced in a way similar to Example III-3. The aggregated cellsjumped over the gap between the culturing surface and the shaft, andobtainment of a ringed three-dimensional tissue body wound around theshaft was confirmed. FIG. 24 illustrates the ringed three-dimensionaltissue body obtained in Example III-4. The resulting ringedthree-dimensional tissue body had ADSC as the principal component.

Example III-5 (Production of Luminal Three-Dimensional Tissue Body)

The production apparatus of a three-dimensional tissue body manufacturedin Example III-1 was placed in a conical tube (product code “2345-050”by Iwaki & Co) with an inner diameter of 30 mm and immersed in a medium(RPMI-1640+10% FBS (lot number 715929 by Biological Industries)+10 ng/μLrat TGF-β1 recombinant (catalog number 100-21 by PeproTech)+50 μg/mL ofascorbic acid diphosphate (catalog number 196-01252 by Wako PureChemical Industries). Subsequently, chondrocytes collected from the kneejoint of a GFP recombinant Lewis rat with a conventional method weremixed into a medium, similar to the medium in which the productionapparatus was immersed, to produce a cell suspension. Next, 2 mL of thecell suspension at 60×10⁵ cells/mL was added to seed cells, and thecells were cultured for 48 hours in a cell incubator (37° C., 5% CO₂).Obtainment of a ringed three-dimensional tissue body wound around theshaft was confirmed.

Subsequently, the culturing surface was moved downward 0.5 mm in theextending direction of the shaft, another 2 mL of the cell suspensionwas added to seed cells again, and the cells were cultured for 48 hoursin a cell incubator (37° C., 5% CO₂) (second cell seeding andculturing). Seeding and culturing of cells in the same way werecontinuously repeated a total of 9 times.

Obtainment of a luminal three-dimensional tissue body in which 9 ringedthree-dimensional tissue bodies were connected and adhered to each otherwas confirmed.

FIG. 25 illustrates the luminal three-dimensional tissue body obtainedin Example III-5. Note that FIG. 25 is a photograph taken afterreplacing the stainless-steel tube shaft with a silicone resin tube.

Example III-6 (Production of Luminal Three-Dimensional Tissue Body)

Except for using the production apparatus of a three-dimensional tissuebody manufactured in Example III-2 and moving the culturing surfaceupward 0.2 mm in the extending direction of the shaft, a luminalthree-dimensional tissue body was produced in a way similar to ExampleIII-5. Obtainment of a luminal three-dimensional tissue body in which 9ringed three-dimensional tissue bodies were connected and adhered toeach other was confirmed.

The resulting luminal three-dimensional tissue body was nearly identicalto the one in FIG. 25.

Example III-7

(Manufacturing of Production Apparatus, of Three-Dimensional TissueBody, with a Plurality of Culturing Surfaces)

A stainless-steel tube (by Fuji Filter Manufacturing Co., 3ϕ) with ameshed surface was used as a shaft, and “PrimeSurface®” (numberMS-9024X, by Sumitomo Bakelite Co.) was used as a culturing surface, aportion of the culturing surface being cut out to form a through hole(approximately 3.2 mm diameter), to manufacture an apparatus with 9culturing surfaces and a gap between the culturing surfaces and theshaft. All of the culturing surfaces were substantially circular, withan outer diameter of approximately 24 mm. The gap between the culturingsurfaces and the shaft was 0.2 mm. The apparatus was shaped as in FIG.26, except with nine culturing surfaces and one shaft passed through thethrough holes of the nine culturing surfaces. As in Example III-1, theculturing surfaces were all coated with a temperature-responsive polymerto prepare a production apparatus, of a three-dimensional tissue body,having coated culturing surfaces.

The gap between culturing surfaces was 1 mm.

Example III-8 (Production of Luminal Three-Dimensional Tissue Body)

The production apparatus of a three-dimensional tissue body manufacturedin Example III-7 was placed in a conical tube (product code “2345-050”by Iwaki & Co) with an inner diameter of 30 mm and immersed in a medium(DMEM+10% FBS (lot number 715929 by Biological Industries)).Subsequently, rat subcutaneous adipose-derived mesenchymal stem cellswere mixed into a medium, similar to the medium in which the productionapparatus was immersed, to produce a cell suspension. Next, 2 mL of thecell suspension at 60×10⁶ cells/mL was added to seed cells on eachcoated culturing surface, and the cells were cultured for 48 hours in acell incubator (37° C., 5% CO₂). Obtainment from each coated culturingsurface of a ringed three-dimensional tissue body wound around the shaftwas confirmed.

Subsequently, culturing was further continued for 48 hours, after whichobtainment of a luminal three-dimensional tissue body, in which 9 ringedthree-dimensional tissue bodies formed from the coated culturingsurfaces were connected and adhered to each other, was confirmed.

FIG. 28A illustrates the luminal three-dimensional tissue body obtainedin Example III-8 (photograph after 3.8% glutaraldehyde fixation), andFIG. 28B illustrates an HE stain section image.

Example III-9 (Synthetic Blood Vessel)

Cells were seeded by mixing vascular endothelial cells, derived fromumbilical cord blood and labeled with a red fluorescent label using acell linker kit, in a medium (DMEM+10% FBS (lot number 715929 byBiological Industries) and adding 1 mL at a time at a concentration of60×10⁵ cells/mL to the nine culturing surfaces of the productionapparatus of a three-dimensional tissue body manufactured in ExampleIII-7. The apparatus was placed in a conical tube (product code“2345-050” by Iwaki & Co) with an inner diameter of 30 mm and immersedin a medium (DMEM+10% FBS (lot number 715929 by Biological Industries)).The cells were then cultured for 48 hours in a cell incubator (37° C.,5% CO₂). Obtainment of a ringed three-dimensional tissue body ofvascular endothelial cells wound around the shaft was confirmed.

Subsequently, GFP knock-in rat subcutaneous adipose-derived mesenchymalstem cells were mixed into a medium, similar to the medium in which theproduction apparatus was immersed, to produce a mesenchymal stem cellsuspension. Ten mL at 60×10⁵ cells/mL was added to seed cells, and thecells were cultured for 48 hours in a cell incubator (37° C., 5% CO₂).Obtainment of a luminal three-dimensional tissue body (synthetic bloodvessel) in which 9 ringed three-dimensional tissue bodies were connectedand adhered to each other, the tissue bodies having a two-layerstructure with a layer of mesenchymal stem cells surrounding a layer ofvascular endothelial cells, was confirmed.

FIG. 29 illustrates a fluorescence microscope image of the syntheticblood vessel obtained in Example III-9.

Example III-10 (Synthetic Blood Trachea)

The production apparatus of a three-dimensional tissue body manufacturedin Example III-1 was placed in a conical tube (product code “2345-050”by Iwaki & Co) with an inner diameter of 30 mm and immersed in a medium(DMEM+10% FBS (lot number 715929 by Biological Industries)+50 μg/mL ofascorbic acid diphosphate (catalog number 196-1252 by Wako Pure ChemicalIndustries)).

Subsequently, beagle knee joint-derived chondrocytes were mixed into amedium, similar to the medium in which the production apparatus wasimmersed, to produce a chondrocyte suspension. Two mL of the chondrocytesuspension at 60×10⁵ cells/mL was then added to seed cells, and thecells were cultured for 48 hours in a cell incubator (37° C., 5% CO₂).Obtainment of a ringed three-dimensional tissue body of chondrocyteswound around the shaft was confirmed.

Subsequently, the culturing surface was moved downward 0.5 mm in theextending direction of the shaft, and 2 mL of a fibroblast suspension(60×10⁵ cells/mL), produced by mixing rat adipose-derived mesenchymalstem cells (ADSC) into a similar medium as the medium in which theproduction apparatus was immersed, was added to seed cells again. Thecells were cultured for 48 hours in a cell incubator (37° C., 5% CO₂)(second cell seeding and culturing).

Chondrocytes and mesenchymal stem cells were used alternately.Obtainment of a luminal three-dimensional tissue body (synthetictrachea) in which 6 ringed three-dimensional tissue bodies wereconnected and adhered to each other was confirmed.

FIG. 30 illustrates the synthetic trachea obtained in Example III-10.Note that FIG. 30 is a photograph taken after replacing thestainless-steel tube shaft with a silicone resin tube.

Example III-11 (Three-Dimensional Tissue Body Having Protein asPrincipal Component)

The production apparatus of a three-dimensional tissue body manufacturedin Example III-1 was placed in a conical tube (product code “2345-050”by Iwaki & Co) with an inner diameter of 30 mm and immersed in a medium(DMEM+10% FBS (lot number 715929 by Biological Industries)+50 μg/mL ofascorbic acid diphosphate (catalog number 196-1252 by Wako Pure ChemicalIndustries)).

Subsequently, rat adipose-derived mesenchymal stem cells (ADSC) weremixed into a medium, similar to the medium in which the productionapparatus was immersed, to produce a cell suspension. Next, 2 mL of thecell suspension at 60×10⁵ cells/mL was added to seed cells, and thecells were cultured for 48 hours in a cell incubator (37° C., 5% CO₂).Obtainment of a ringed three-dimensional tissue body wound around theshaft was confirmed.

Subsequently, the culturing surface was moved downward 0.5 mm in theextending direction of the shaft, another 2 mL of the cell suspensionwas added to seed cells again, and the cells were cultured for 48 hoursin a cell incubator (37° C., 5% CO₂) (second cell seeding andculturing).

Seeding and culturing of cells in the same way was continuously repeateda total of 6 times.

Obtainment of a luminal three-dimensional tissue body in which 6 ringedthree-dimensional tissue bodies were connected and adhered to each otherwas confirmed.

Subsequently, the obtained luminal three-dimensional tissue body wastreated with sodium dodecyl sulfate and ethanol and then treated with3.8% glutaraldehyde to kill the cells in the three-dimensional tissuebody. Obtainment of a luminal three-dimensional tissue body havingprotein as the principal component was confirmed.

FIG. 31 illustrates the three-dimensional tissue body, obtained inExample III-11, having protein as the principal component.

Examples of Aspect (IV)

Aspect (IV) is described below in greater detail with reference toexamples, but the present disclosure is in no way limited by theseexamples.

In the following tests, commercially available reagents were usedwithout further purification, unless otherwise noted.

Example IV-A1

(Test IV-A1) Manufacturing of Polymer

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and 5 mLof water were added to a 50 mL capacity transparent vial made of softglass, and the vial was stirred using a magnetic stirrer. The mixture(liquid) was then purged with Gi-grade, highly pure (purity: 99.99995%)nitrogen gas for 10 minutes (flow rate: 2.0 L/min) to deoxygenize themixture. The DMAEMA that was used included 0.5 mass % ofmethylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain atemperature-responsive (homo)polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=1.0×10⁵ g/mol (Mw/Mn=10.0) (Example Polymer 1).

The nuclear magnetic resonance (NMR) spectrum of the above-describedtemperature-responsive polymer was measured using a nuclear magneticresonance apparatus (model Gemini-300 by Varian) with heavy water (D₂O)as a standard substance. A representative peak is indicated below.

¹H-NMR (in D₂O) δ 0.8-1.2 (br, —CH₂—C(CH₃)—), 1.6-2.0 (br,—CH₂—C(CH₃)—), 2.2-2.4 (br, —N(CH₃)₂), 2.5-2.7 (br, —CH₂—N(CH₃)₂),4.0-4.2 (br, —O—CH₂—).

Here, from the number of protons A in the methyl group (δ 0.8-1.2) ofthe main chain (3 per monomer molecule in the case of a DMAEMAhomopolymer) and the number of methyl protons B in the dimethylaminogroup (δ 2.2-2.4) of the side chain (6 per monomer molecule in the caseof a DMAEMA homopolymer), the ratio was calculated between the number offunctional groups that are amino groups in the side chain and the numberof functional groups that are carboxyl groups in the side chain producedby a hydrolysis reaction, which proceeds simultaneously with thepolymerization reaction, of an ester bond of the side chain.

The resulting ratio was 94:6 in the case of the above-describedtemperature-responsive polymer. Converting into the C/A ratio for an ioncomplex in a two-component mixed system that includes a cationic polymerand an anionic polymer yields a C/A ratio of 15.6.

A 3% aqueous solution of Example Polymer 1 was produced, and theabsorbance of the aqueous solution at 660 nm was measured between 20° C.and 40° C.

Between 20° C. and 30° C., the aqueous solution was transparent, with anabsorbance of nearly 0. Starting around 31° C., however, the aqueoussolution became cloudy, and the absorbance increased suddenly at 32° C.The aforementioned polymer was thus confirmed to have a cloud point ofapproximately 32° C.

Once the Example Polymer was increased in temperature to 37° C., thepolymer aqueous solution was suspended with good responsiveness.Subsequently, the entire aqueous solution hardened. When maintained atroom temperature (25° C.), the hardened product retained its hard statefor several tens of hours. Subsequently, the hardened product graduallydissolved, changing into a homogeneous aqueous solution. Upon beingcooled to 4° C., the hardened polymer rapidly dissolved. Repeating theaforementioned operation to raise and lower the temperature caused nochange in responsiveness, thereby confirming that the polymer reversiblyunderwent phase transitions.

(Test IV-A2) Manufacturing of Cell Structure

In Example IV-A1, a cell structure was manufactured using a culturingsurface produced by application and drying.

A 35 mm polystyrene cell culture plate (model 3000-035-MYP by Iwaki &Co., Ltd, bottom area of 9 cm² per well) was used as a cell culturecontainer.

Next, 40 μL of a temperature-responsive polymer aqueous solution(concentration: 15 μg/mL) cooled to the cloud point or below was appliedover the entire culturing surface.

The applied aqueous solution of temperature-responsive polymer was driedby leaving the cell culture plate on a clean bench.

A coated region that was coated with the temperature-responsive polymerwas thus provided on the culturing surface of the cell culture plate.

(Test IV-A3) Measurement of Zeta Potential

The surface zeta potential of the coated region, which was provided on asmall piece of a cell culture plate with a procedure similar to theprocedure for Test IV-A2, was measured using a zeta potential meter(model ELSZ by Otsuka Electronics Co., Ltd) and a cell unit for flatplate samples.

Specifically, a sample of the small piece was tightly adhered to thebottom surface of a quartz cell, and a monitor particle suspension wasinjected into the cell. Here, particles (zeta potential: −5 mV to +5 mV)yielded by coating polystyrene latex (particle size: approximately 500nm) with hydroxypropyl cellulose (Mw=30,000) were used as standardmonitoring particles. A 10 mM sodium chloride aqueous solution at pH=7and 37° C. was used as a solvent. The zeta potential was calculatedusing the Smoluchowski equation.

The zeta potential of the surface of a small piece of non-coated cellculture plate was −68 mV, which is a value known to a person skilled inthe art as the zeta potential of a solid surface of a typicalthermoplastic resin.

In contrast, the zeta potential of the surface of the small piece ofcell culture plate coated with a temperature-responsive polymer was +20mV.

As a person skilled in the art knows, the measured value of the zetapotential of a solid surface exhibits approximately ±10% variation withcurrent techniques. Variation is also present in the coating operationitself during the step of producing the sample. Hence, theaforementioned measurement of the zeta potential may have a certainerror.

(Test IV-A4) Measurement of Contact Angle

The contact angle of water relative to the coated region of the cellculture plate was measured as 700±10° using a contact angle meter(DMs-400, by Kyowa Interface Science Co.) in conformity with JIS R3257.

(Test IV-A5) Cell Culture

Here, 3.0×10⁵ cells/mL of mesenchymal adipose stem cells, derived fromrat subcutaneous fat and tagged with GFP, were suspended in a completemedium (Dulbecco's Modified Eagle Medium (DMEM)+10% fetal bovine serum(FBS); DMEM: model 11995-065 by Gibco; FCS: lot number 928696, byInvitrogen) to produce a cell suspension.

Droplets of the cell suspension were spotted with a pipettor in 100locations (see (iv) of FIG. 32) while appropriately selecting betweenamounts of 0.5 μL, 2 μL, 4 μL, and 20 μL. The shape of the droplets wasnearly a true circle, and the diameter of the droplets was 1 mm in thecase of 0.5 μL, 2 mm in the case of 2 μL, 3 mm in the case of 4 μL, and5.5 mm in the case of 20 μL. The gap between any two droplets was 200 μmor more. The ratio of the bottom area of the droplet to the area of thecoated region was approximately 75%.

The seeded adipose stem cells derived from rat subcutaneous fat werecultured for 8 hours in a cell culture incubator at 37° C. in a 5% CO₂atmosphere.

At 0 hours after the start of culturing (immediately after spotting),the adipose stem cells were adhered to the entire coated region (see (v)of FIG. 32).

At 2 hours after the start of culturing, the cells located near theperiphery of the coated region spontaneously started to peel off.

From 3 hours to 6 hours after the start of culturing, spontaneousdetachment of cells progressed slowly from the periphery of the coatedregion towards the center of the coated region.

At 8 hours after the start of culturing, cell structures having aspheroidal structure finally formed from the cultures in the coatedregions, the cell structures being equal in number to the number ofcoated regions (see FIG. (vi) of FIG. 32).

The formed spheroidal cell structures uniformly had a nearly sphericalshape with a desired size in accordance with the amount of the droplet.

Examples IV-A2 to IV-A5

In Example IV-A2, an experiment was performed in a way similar toExample IV-A1, except for changing the surface zeta potential of thecoated region from +20 mV to +15 mV by adjusting the conditions formanufacturing the polymer. Cell structures with a spheroidal structureand a uniform shape of a desired size were obtained, as in ExampleIV-A1.

In Example IV-A3, an experiment was performed in a way similar toExample IV-A1, except for changing the surface zeta potential of thecoated region from +20 mV to +35 mV by adjusting the conditions formanufacturing the polymer. Cell structures with a spheroidal structureand a uniform shape of a desired size were obtained, as in ExampleIV-A1.

In Example IV-A4, an experiment was performed in a way similar toExample IV-A1, except for changing the contact angle of water relativeto the coated region from 70°±10° to 65°±7° by adjusting the conditionsfor manufacturing the polymer. Cell structures with a spheroidalstructure and a uniform shape of a desired size were obtained, as inExample IV-A1.

In Example IV-A5, an experiment was performed in a way similar toExample IV-A1, except for changing the contact angle of water relativeto the coated region from 70°±10° to 75°±5° by adjusting the conditionsfor manufacturing the polymer. Cell structures with a spheroidalstructure and a uniform shape of a desired size were obtained, as inExample IV-A1.

Example IV-B1

(Test IV-B1) Production of Culturing Surface

A precision glass plate produced by Matsunami Glass Ind., MICRO COVERGLASS (30 mm×40 mm×0.15 mm thick) was washed with diethyl ether anddried. The washing was intended to remove foreign materials and an oilcomponent that prevents glass plates from sticking together.

(Test IV-B1-1) <Case of Entire Culturing Surface being Coated withPolymer>

Vinyltrimethoxysilane was dissolved in a 4% acetic acid aqueous solutionand adjusted to a final concentration of 0.5% or 2.0%. This silanecompound solution was cast over the entire surface of the glass anddrained to yield a solution thickness of approximately 0.1 μm. The glassplate was left to stand for 72 hours in a 25° C. sealed desiccatorincreased to a humidity of 84% with a saturated solution of potassiumchloride. After washing with RO water, the glass was treated for 10minutes at 100° C. to evaporate moisture and was then further washedwith RO water and 2-propanol. It was confirmed with IR analysis thatvinyl groups derived from the silane coupling agentVinyltrimethoxysilane were fixed to the glass surface.

A solution with a mixture of 10 g of2-(N,N-Dimethylaminoethyl)methacrylate and 5 g of RO was bubbled for 10minutes with nitrogen gas. The glass plate, with introduced vinylgroups, produced in (1) above was placed in a 100 mm diameter Petridish, 10 mL of a monomer aqueous solution deoxygenated by nitrogen gasbubbling was added to soak the glass plate, and the Petri dish wassealed to form a nitrogen atmosphere inside. A 375 nm black light wasirradiated from the bottom of the Petri dish for 10 hours, and the glassplate was washed with RO water and 2-propanol and dried to yield a cellculture container (1).

The correlation between the droplet amount and the droplet diameter inthe cell culture container (1) produced in Test IV-B1-1 wasinvestigated.

With a pipettor, 0.5 μL to 50 μL droplets of a rat subcutaneousadipose-derived mesenchymal stem cell suspension (cell density: 3.0×10⁵cells/mL, complete medium (Dulbecco's Modified Eagle Medium (DMEM)+10%fetal bovine serum (FBS) solution; DMEM: model 11995-065 by Gibco; FCS:lot number 928696, by Invitrogen)) were spotted on the glass platesurface to which the copolymer was fixed. The diameter of the dropletswas then measured from a projection image of the bottom. The dropletamount and the droplet diameter did not have a first order correlationover the entire range of the droplet amounts of 0.5 μL to 50 μL, asillustrated in FIG. 34A.

On the other hand, dividing into a 0.5 μL to 5.0 μL range and a 5.0 μLto 50 μL range, as illustrated in FIGS. 34B and 34C, revealed a linearcorrelation in each region and suggested that the droplet diameter canbe controlled by the droplet amount.

FIGS. 34A to 34C illustrate the results of investigating the correlationbetween the amount of a droplet and the diameter of the droplet on theculturing surface in an example.

(Test IV-B1-2) <Case of Coating a Plurality of Locations on theCulturing Surface with Polymer>

Vinyltrimethoxysilane was dissolved in a 4% acetic acid aqueous solutionand adjusted to a final concentration of 2.0%. Twenty droplets of thesilane coupling agent solution were spotted 0.5 μL at a time onto aprecision glass plate, produced by Matsunami Glass Ind., in four rowsand five columns at intervals of 1.2 mm to 12 mm starting from thecenter of the droplet. The glass plate was left to stand for 72 hours ina 25° C. desiccator increased to a humidity of 84% with a saturatedsolution of potassium chloride. The droplets were suctioned with acapillary, and the glass plate was washed with water and methanol anddried to yield a cell culture container (2).

As in Test IV-B1-1, graft polymerization was performed. It was confirmedwith IR analysis that a copolymer was fixed only onto the droplets(dots) where the silane coupling agent Vinyltrimethoxysilane wasspotted.

(Test IV-B2) Manufacturing of Cell Structure

In Example IV-B2, a cell structure was manufactured using a culturingsurface produced by graft polymerization.

(Test IV-B2-1)

With a pipettor, 20 droplets of a rat subcutaneous adipose-derivedmesenchymal stem cell suspension (cell density: 3.0×10⁵ cells/mL,complete medium (Dulbecco's Modified Eagle Medium (DMEM)+10% fetalbovine serum (FBS) solution; DMEM: model 11995-065 by Gibco; FCS: lotnumber 928696, by Invitrogen)) were spotted in a range of 0.5 μL to 2.0μL in four rows and five columns on the glass plate surface,manufactured in Test IV-B1-1, to which the copolymer was fixed. Thecells were then cultured in a cell culture incubator (MCO-5C by SanyoElectric Co.) for 24 hours (37° C., 5% carbon dioxide gas).

Individual cells in the droplet formed a single layer and adhered to theglass surface, and a state of cells accumulating on top of other cellswas hardly observed. One hour after spotting of the droplets, all of thecells were adhered to the glass plate and were expanding. Afterapproximately 9 hours of further culturing, cells began to aggregatefrom the periphery of the droplet towards the center, and after 21hours, all of the cells had gathered in one place to form a spheroidalmass at the central portion.

After soaking the glass plate in a 0.3% methyl cellulose PBS solution,all of the spheroids could be collected as a solution with suspendedspheroids.

Droplets fused when the distance between them was 30 μm or less,possibly due to attraction or the surface tension relative to the airphase. Consequently, a state in which a suspension was cast over theentire glass plate was reached, resulting in cells simultaneouslyaggregating everywhere.

Examples of Aspect (V)

Aspect (V) is described below in greater detail with reference toexamples, but the present disclosure is in no way limited by theseexamples.

In the following tests, commercially available reagents were usedwithout further purification, unless otherwise noted.

Example V-1

(Test V-A) Production of Temperature-Responsive Polymer

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and5,000 μL of water were added to a 50 mL capacity transparent vial madeof soft glass, and the vial was stirred using a magnetic stirrer. Themixture (liquid) was then purged with Gi-grade, highly pure (purity:99.99995%) nitrogen gas for 10 minutes (flow rate: 2.0 L/min) todeoxygenize the mixture. The DMAEMA that was used included 0.5 weight %of methylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain an intramolecular ioncomplex-type temperature-responsive polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=100,000 (Mw/Mn=10.0).

The nuclear magnetic resonance (NMR) spectrum of thetemperature-responsive polymer was measured using a nuclear magneticresonance apparatus (model Gemini-300 by Varian) with heavy water (D₂O)as a standard substance. The representative peaks common to ExamplePolymer V-1 are listed below.

¹H-NMR (in D₂O) δ 0.8-1.2 (br, 3H, —CH₂—C(CH₃)—), 1.6-2.0 (br, 2H,—CH₂—C(CH₃)—), 2.2-2.4 (br, 6H, —N(CH₃)₂), 2.5-2.7 (br, 1.9H,—CH₂—N(CH₃)₂), 4.0-4.2 (br, 1.9H, —O—CH₂—).

Here, from the number of protons A in the methyl group (δ 0.8-1.2)bonded at the a position (3 in both the case of a DMAEMA unit and thecase of a methacrylic acid unit) and the number of methyl protons B inthe ethyl group (δ 4.0-4.2) bonded to oxygen in the ester bond of a sidechain (2 in the case of a DMAEMA unit and 0 in the case of a methacrylicacid unit), the ratio between the number of functional groups that areamino groups in the side chains of DMAEMA and the number of functionalgroups that are carboxyl groups in the side chains of methacrylic acidwas calculated.

The resulting ratio was 94:6 in the case of the obtained polymer V-1.Converting into the C/A ratio for an ion complex in a two-componentmixed system that includes a cationic polymer and an anionic polymeryields a C/A ratio of 15.6.

Furthermore, a 3% aqueous solution of the obtainedtemperature-responsive polymer was produced, and measuring theabsorbance of the aqueous solution at 660 nm between 20° C. and 40° C.yielded a value of approximately 32° C.

(Test V-B) Preparation of First Coated Regions and Second Coated Regions

A ϕ35 mm low cell-adsorption plate (PrimeSurface® by SUMILON) was usedas a cell culture container.

At room temperature, 4.0 μL at a time of the above-described polymeraqueous solution produced in Test V-A (concentration: 15 ng/μL) wasspotted onto 6 locations on the culturing surface of the aforementionedplate. The aqueous solution was then dried for 30 minutes at 37° C. toprepare circular first coated regions (area 4.5 mm²).

After drying, 0.2 μL at a time of a fibronectin solution (derived fromhuman plasma) (concentration: 200 ng/μL) (lot number 3353563 by BDBiosciences) was spotted onto 1 of the 6 circular first coated regions,at 2 locations positioned on the edge of the first coated region along aline passing through the center of the first coated region, so as tooverlap the first coated region. The solution was then dried at 37° C.for 5 minutes to prepare circular second coated regions (area 0.8 mm²)(see FIG. 38A).

The surface zeta potential of the first coated region was measured usinga zeta potential meter (model ELSZ by Otsuka Electronics Co.) and a cellunit for flat plate samples. During the measurement, a quartz cell wasused as the cell, a particle (zeta potential: −5 mV to +5 mV) in whichpolystyrene latex (particle size: approximately 500 nm) was coated withhydroxypropyl cellulose (Mw=30,000) was used as a reference monitorparticle, and a 10 mM sodium chloride aqueous solution at pH=7 and 37°C. was used as a solvent. The zeta potential was calculated with theSmoluchowski equation.

The resulting zeta potential of the surface of the first coated regioncoated with a temperature-responsive polymer was +20 mV. As is wellknown to a person skilled in the art, the measured value of the zetapotential exhibits a variation of approximately ±10%.

The contact angle of water relative to the first coated region of a cellculture plate was measured as 700±10° using a contact angle meter(DMs-400, by Kyowa Interface Science Co.) in conformity with JIS R3257.

(Test V-C) Seeding and Culturing of Cells

The cell culture container prepared in Test V-B was used.

Adipose-derived mesenchymal stem cells (adipose-derived vascular stromalcells (ADSC)) of a GFP recombinant Lewis rat were suspended in a medium(Dulbecco's Modified Eagle Medium (DMEM)+10% fetal bovine serum (FBS);DMEM: model 11965 by Gibco; FBS: lot number 715929 by BiologicalIndustries) to produce a cell suspension.

The cell suspension was added to the plate at room temperature to yielda cell density of at least 2.5×10⁵ cells/cm² (95% confluency).

These cells were then cultured for 2 hours in a cell culture incubatorat 37° C. and 5% CO₂.

At 2 hours after the start of culturing, the medium was exchanged withthe above-described medium, and dead cells were removed.

FIG. 38A is a photograph when using a microscope, in Test V-C, toobserve the state after 2 hours of culturing ADSC of a GFP recombinantLewis rat in the first coated region and the second coated regionsprepared in Test V-B.

The cells were then further cultured for 18 hours in a cell cultureincubator at 37° C. and 5% CO₂.

During these 18 hours of culturing, the cells adhered to the firstcoated region aggregated towards the central portion, whereas the cellsadhered to the second coated regions remained adhered to their initialposition. The bonding strength of the inter-cell network resulting frommaturation of seeded cells exceeded the adhesiveness of cells to thefirst coated region, leading to cell aggregation. At this time,aggregation and contraction in the long axis direction was limited,whereas aggregation and contraction in the short axis direction wasprioritized. Ultimately, a linear cell structure was formed from the 2cell groups that remained adhered to the second coated regions and thestick-like cell group connecting the 2 cell groups.

FIG. 38B is a photograph when using a microscope, in Test V-C, toobserve the state after 20 hours of culturing ADSC of a GFP recombinantLewis rat in the first coated region and the second coated regionprepared in Test V-B.

FIG. 38C is a photograph when observing the cell structure in FIG. 38Bat lower magnification.

FIG. 38D is a photograph when using a fluorescence microscope to observethe state of the cell structure indicated by the dashed line in FIG.38B. It is clear that within the cell structure obtained in Test V-C,the stick-like cell group had a shape such that cells spread in theextending direction of the cell structure, and the stick-like cell groupwas oriented in the direction of a line connecting the 2 cell groupsthat remained adhered to the second coated regions.

Example V-2

The cell adhesive substance that was used was changed from a fibronectinsolution to laminin (concentration: 50 ng/μL) (by Nippi, Inc.), and asimilar test to the above-described Test V-B of Example V-1 wasperformed. Also, the cells that were used were changed from ADSC of aGFP recombinant Lewis rat to cardiomyocytes (separated from a newbornLewis rat on the first day after birth with a conventional method), anda similar test to the above-described Test V-C of Example V-1 wasperformed.

In this case as well, a cell structure with a structure similar to thatof the above-described cell structure indicated in FIGS. 38A to 38D wasobtained.

TABLE 4 Example V-1 Example V-2 Preparation First Coated Shape circularcircular Step Region Area (mm²) 4.5 4.5 Second Shape circular circularCoated Area (mm²) 0.8 0.8 Regions Number 2 2 Cell Adhesive fibronectinlaminin Substance Coated 40 10 Amount (ng) Cell Non-Adhesive Wall nonenone Seeding and Cells Type ADSC cardio- Culturing myocytes Step Density2.5 × 10⁵ 2.5 × 10⁵ (cells/cm²) Cell Shape spindle spindle StructureFigure FIGS. 38A FIGS. 38A to 38D to 38D

Examples of Aspect (VI)

Aspect (VI) is described below in greater detail with reference toexamples, but the present disclosure is in no way limited by theseexamples.

Example VI-1

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and 5 mLof water were added to a 50 mL capacity transparent vial made of softglass, and the vial was stirred using a magnetic stirrer. The mixture(liquid) was then purged with Gi-grade, highly pure (purity: 99.99995%)nitrogen gas for 10 minutes (flow rate: 2.0 L/min) to deoxygenize themixture. The DMAEMA that was used included 0.5 mass % of methylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain atemperature-responsive (homo)polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=1.0×10⁵ g/mol (Mw/Mn=10.0).

The nuclear magnetic resonance (NMR) spectrum of the above-describedtemperature-responsive polymer was measured using a nuclear magneticresonance apparatus (model Gemini-300 by Varian) with heavy water (D₂O)as a standard substance. A representative peak is indicated below.

¹H-NMR (in D₂O) δ 0.8-1.2 (br, —CH₂—C(CH₃)—), 1.6-2.0 (br,—CH₂—C(CH₃)—), 2.2-2.4 (br, —N(CH₃)₂), 2.5-2.7 (br, —CH₂—N(CH₃)₂),4.0-4.2 (br, —O—CH₂—).

Here, from the number of protons A in the methyl group (δ 0.8-1.2) ofthe main chain (3 per monomer molecule in the case of a DMAEMAhomopolymer) and the number of methyl protons B in the dimethylaminogroup (δ 2.2-2.4) of the side chain (6 per monomer molecule in the caseof a DMAEMA homopolymer), the ratio was calculated between the number offunctional groups that are amino groups in the side chain and the numberof functional groups that are carboxyl groups in the side chain producedby a hydrolysis reaction, which proceeds simultaneously with thepolymerization reaction, of an ester bond of the side chain.

The resulting ratio was 94:6 in the case of the above-describedtemperature-responsive polymer. Converting into the C/A ratio for an ioncomplex in a two-component mixed system that includes a cationic polymerand an anionic polymer yields a C/A ratio of 15.6.

The cloud point of the above-described temperature-responsive polymerwas measured with the following method.

A 3% aqueous solution of the temperature-responsive polymer wasproduced, and the absorbance of the aqueous solution at 660 nm wasmeasured between 20° C. and 40° C.

Between 20° C. and 30° C., the aqueous solution was transparent, with anabsorbance of nearly 0. Starting around 31° C., however, the aqueoussolution became cloudy, and the absorbance increased suddenly at 32° C.The temperature-responsive polymer was thus confirmed to have a cloudpoint of approximately 32° C.

Once the temperature-responsive polymer was increased in temperature to37° C., the polymer aqueous solution was suspended with goodresponsiveness. Subsequently, the entire aqueous solution hardened. Whenmaintained at room temperature (25° C.), the hardened product retainedits hard state for several tens of hours. Subsequently, the hardenedproduct gradually dissolved, changing into a homogeneous aqueoussolution. Upon being cooled to 4° C., the hardened polymer rapidlydissolved. Repeating the aforementioned operation to raise and lower thetemperature caused no change in responsiveness, thereby confirming thatthe polymer reversibly underwent phase transitions.

The above-described temperature-responsive polymer was dissolved in purewater to produce a temperature-responsive polymer solution (finalconcentration 6 ng/μL). Next, 0.5 μL of the temperature-responsivepolymer solution was applied in circles to 10 locations on the culturingsurface of a 35 mm dish (“PrimeSurface®”, MS-9035X, by Sumitomo BakeliteCo.).

The diameter of the circular coated culturing surface at each locationwas approximately 2,000 μm, and the distance between coated culturingsurfaces was approximately 2,000 μm.

The applied temperature-responsive polymer aqueous solution was thendried by leaving the dish in an incubator (40° C.) for 1 hour to preparea coated cell culture container having a plurality of coated culturingsurfaces.

Mixed cells, in which newborn Lewis rat cardiomyocytes andadipose-derived mesenchymal stem cells (ADSC) of a GFP recombinant Lewisrat were mixed at a ratio of 300 mesenchymal stem cells per 100cardiomyocytes, were mixed into a medium (SkGM (model CC-3245 by LonzaCo., Ltd.)+10% FBS (lot number 715929 by Gibco)) to produce a 7.2×10⁶cells/mL mixed cell solution, 5 mL of which was added to a coated cellculture container to seed cells.

Subsequently, cells were cultured for 1 hour in a cell incubator (37°C., 5% CO₂), and the medium was exchanged using a new medium. The cellswere then further cultured for 48 hours in a cell incubator (37° C., 5%CO₂) to obtain an aggregated cell structure.

Example VI-2

Except for mixing newborn Lewis rat cardiomyocytes and adipose-derivedmesenchymal stem cells of a GFP recombinant Lewis rat at a ratio of 233mesenchymal stem cells per 100 cardiomyocytes, an aggregated cellstructure was obtained in a way similar to Example VI-1.

Example VI-3

Three types of cells were used: newborn Lewis rat cardiomyocytes ascardiomyocytes, adipose-derived mesenchymal stem cells (ADSC) of a GFPrecombinant Lewis rat as mesenchymal stem cells, and macrophages asimmune system cells. The macrophages were used after inducingdifferentiation from monocytes derived from rat bone marrow (numberBMM01, melomonocyte culturing kit, by Cosmo Bio).

As the first stage, newborn Lewis rat cardiomyocytes and adipose-derivedmesenchymal stem cells of a GFP recombinant Lewis rat were mixed at aratio of 250 mesenchymal stem cells per 100 cardiomyocytes, the mixedcells were seeded in a way similar to Example VI-1, and after 5 hours,the cells had adhered to form a single layer. The medium was thenexchanged. A rat derived macrophage suspension including macrophages wasadded onto the single layer to adjust the ratio to 5 rat derivedmacrophages per 100 newborn Lewis rat cardiomyocytes, and staticculturing was continued. Other than these differences, operationssimilar to Example VI-1 were performed.

When a single culture layer composed of cardiomyocytes and mesenchymalstem cells aggregated like a drawstring bag, the macrophages that hadprecipitated onto the single layer culture layer were incorporatedinside the aggregation, and one aggregated cell structure was obtained.No macrophages were confirmed on the exposed culturing surface, and itwas confirmed that all of the seeded macrophages were incorporated inthe cell aggregation.

Comparative Example VI-1

Except for mixing newborn Lewis rat cardiomyocytes and adipose-derivedmesenchymal stem cells of a GFP recombinant Lewis rat at a ratio of 75mesenchymal stem cells per 100 cardiomyocytes, an aggregated cellstructure was obtained in a way similar to Example VI-1.

Comparative Example VI-2

Except for producing a cell solution composed only of newborn Lewis ratcardiomyocytes, without mixing in adipose-derived mesenchymal stem cellsof a GFP recombinant Lewis rat, an aggregated cell structure wasobtained in a way similar to Example VI-1.

Comparative Example VI-3

Except for mixing newborn Lewis rat cardiomyocytes and adipose-derivedmesenchymal stem cells of a GFP recombinant Lewis rat at a ratio of 33mesenchymal stem cells per 100 cardiomyocytes, an aggregated cellstructure was obtained in a way similar to Example VI-1.

Comparative Example VI-4

Except for mixing newborn Lewis rat cardiomyocytes and adipose-derivedmesenchymal stem cells of a GFP recombinant Lewis rat at a ratio of 25mesenchymal stem cells per 100 cardiomyocytes, an aggregated cellstructure was obtained in a way similar to Example VI-1.

Comparative Example VI-5

Except for mixing newborn Lewis rat cardiomyocytes and adipose-derivedmesenchymal stem cells of a GFP recombinant Lewis rat at a ratio of 100mesenchymal stem cells per 100 cardiomyocytes, an aggregated cellstructure was obtained in a way similar to Example VI-1.

[Evaluation]

(Appearance of Cell Structure)

The aggregated cell structures obtained in the Examples and ComparativeExamples were observed with a microscope (ECLIPSE-Ti by NikonCorporation), and the appearance of the cells was evaluated with thefollowing criteria.

G (good): a spherical aggregated cell structure was obtained on all 10of the coated culturing surfaces.P (poor): on one or more of the 10 coated culturing surfaces, the cellsthat adhered to the coated culturing surface remained as a single layerwithout aggregating, or a cell structure had a warped edge with acombination of an aggregated portion and an adhered portion not peeledoff from the coated culturing surface.

(Occurrence of Beating)

The aggregated cell structures obtained in the Examples and ComparativeExamples were observed with a microscope (ECLIPSE-Ti by NikonCorporation) over a period of 48 hours. The occurrence of beating wasthen evaluated with the following criteria.

G (good): during the 48-hour observation period, beating was initiallyconfirmed but subsequently stopped being observed.P (poor): during the 48-hour observation period, beating was confirmedat every observation.

TABLE 5 Comparative Comparative Comparative Comparative ComparativeExample Example Example Example Example Example Example Example VI-1VI-2 VI-3 VI-1 VI-2 VI-3 VI-4 VI-5 Culture Cell Culture Container 35 mm35 mm 35 mm 35 mm 35 mm 35 mm 35 mm 35 mm Container dish dish dish dishdish dish dish dish Preparation Area of Culturing 900 900 900 900 900900 900 900 Step Surface of Cell Culture Container (mm²) Area of EachCoated 3.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14 Culturing Surface (mm²)Amount of Temperature- 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96Responsive Polymer per Unit Area (ng/mm²) Seeding Number of Fibroblasts300 233 250 75 0 33 25 100 Step per 100 Cardiomyocytes Immune System nono yes no no no no yes Cells Added Number of Immune — — 5 — — — — —System Cells per 100 Cardiomyocytes Percentage of 25 30 28 57 100 75 8050 Cardiomyocytes among Total Number of Seeded Cells (%) Percentage ofFibroblasts 75 70 70 53 0 25 20 50 among Total Number of Seeded Cells(%) Culturing Culturing Temperature 37° C. 37° C. 37° C. 37° C. 37° C.37° C. 37° C. 37° C. Step Culturing Time (hours) 48 48 48 48 48 48 48 48Evaluation Appearance of Cell G G G G P P P P Structure Beating G G G PP P P P

It is clear from Table 5 that the cell structures of the Examples werebeating immediately after cell structure formation, but that beating wassubsequently not observed. The cell structures of the Examples were thusjudged to be useable as a heart disease model. Conversely, the cellstructures of the Comparative Examples were observed to be beatingthroughout the 48-hour observation period and were judged to have a formclose to that of a healthy heart.

Examples of Aspect (VII)

Aspect (VII) is described below in greater detail with reference toexamples, but the present disclosure is in no way limited by theseexamples.

Example VII-1

First, 10.0 g of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA) and 5 mLof water were added to a 50 mL capacity transparent vial made of softglass, and the vial was stirred using a magnetic stirrer. The mixture(liquid) was then purged with Gi-grade, highly pure (purity: 99.99995%)nitrogen gas for 10 minutes (flow rate: 2.0 L/min) to deoxygenize themixture. The DMAEMA that was used included 0.5 mass % ofmethylhydroquinone (MEHQ), which is a polymerization inhibitor.

Subsequently, this reactant was polymerized by irradiation withultraviolet light for 22 hours using a round, black fluorescent lamp(model FCL20BL, 18 W, by NEC Corporation). The reactant became viscous 5hours later and hardened 15 hours later. A polymer was thus obtained asa reaction product. This reaction product was dissolved in 2-propanol,and the solution was transferred to a dialysis tube. Dialysis wasperformed for 72 hours to purify the reaction product.

The solution including the reaction product was filtered with a 0.2 μmcellulose mixed-ester filter (model 25AS020 by Toyo Roshi Kaisha), andthe resulting filtrate was freeze dried to obtain atemperature-responsive (homo)polymer (6.8 g yield, 68% conversionratio). The number-average molecular weight (Mn) of this polymer wasmeasured using a GPC (model LC-10vp series by Shimadzu Corporation) withpolyethylene glycol (TSK series by Shodex) as a standard substance andwas determined to be Mn=1.0×10⁵ g/mol (Mw/Mn=10.0).

The nuclear magnetic resonance (NMR) spectrum of the above-describedtemperature-responsive polymer was measured using a nuclear magneticresonance apparatus (model Gemini-300 by Varian) with heavy water (D₂O)as a standard substance. A representative peak is indicated below.

¹H-NMR (in D₂O) δ 0.8-1.2 (br, —CH₂—C(CH₃)—), 1.6-2.0 (br,—CH₂—C(CH₃)—), 2.2-2.4 (br, —N(CH₃)₂), 2.5-2.7 (br, —CH₂—N(CH₃)₂),4.0-4.2 (br, —O—CH₂—).

Here, from the number of protons A in the methyl group (δ 0.8-1.2) ofthe main chain (3 per monomer molecule in the case of a DMAEMAhomopolymer) and the number of methyl protons B in the dimethylaminogroup (δ 2.2-2.4) of the side chain (6 per monomer molecule in the caseof a DMAEMA homopolymer), the ratio was calculated between the number offunctional groups that are amino groups in the side chain and the numberof functional groups that are carboxyl groups in the side chain producedby a hydrolysis reaction, which proceeds simultaneously with thepolymerization reaction, of an ester bond of the side chain.

The resulting ratio was 94:6 in the case of the above-describedtemperature-responsive polymer. Converting into the C/A ratio for an ioncomplex in a two-component mixed system that includes a cationic polymerand an anionic polymer yields a C/A ratio of 15.6.

The cloud point of the above-described temperature-responsive polymerwas measured with the following method.

A 3% aqueous solution of the temperature-responsive polymer wasproduced, and the absorbance of the aqueous solution at 660 nm wasmeasured between 20° C. and 40° C.

Between 20° C. and 30° C., the aqueous solution was transparent, with anabsorbance of nearly 0. Starting around 31° C., however, the aqueoussolution became cloudy, and the absorbance increased suddenly at 32° C.The temperature-responsive polymer was thus confirmed to have a cloudpoint of approximately 32° C.

Once the temperature-responsive polymer was increased in temperature to37° C., the polymer aqueous solution was suspended with goodresponsiveness. Subsequently, the entire aqueous solution hardened. Whenmaintained at room temperature (25° C.), the hardened product retainedits hard state for several tens of hours. Subsequently, the hardenedproduct gradually dissolved, changing into a homogeneous aqueoussolution. Upon being cooled to 4° C., the hardened polymer rapidlydissolved. Repeating the aforementioned operation to raise and lower thetemperature caused no change in responsiveness, thereby confirming thatthe polymer reversibly underwent phase transitions.

The above-described temperature-responsive polymer was dissolved in purewater to produce a temperature-responsive polymer solution (finalconcentration 15 ng/μL). Next, 0.5 μL of the temperature-responsivepolymer solution was applied in circles to 8 locations on the culturingsurface of a 35 mm dish (“PrimeSurface®”, MS-9035X, by Sumitomo BakeliteCo.).

The diameter of the circular coated culturing surface at each locationwas approximately 2,000 μm, and the distance between coated culturingsurfaces was approximately 2,500 μm.

The applied temperature-responsive polymer aqueous solution was thendried by leaving the dish in an incubator (37° C.) for 30 minutes toprepare a coated cell culture container having a plurality of coatedculturing surfaces.

Human hepatoma cells (number “HepG2-500” by Cosmo Bio) were stainedusing a PKH26 Red Fluorescent Cell Linker Kit (by Sigma). Mixed cells,in which the stained hepatoma cells and adipose-derived mesenchymal stemcells (ADSC) of a GFP recombinant Lewis rat were mixed at a ratio of 33mesenchymal stem cells per 100 hepatoma cells, were mixed into a medium(DMAEM+10% FBS (lot number 715929 by Gibco)) to produce a 3.2×10⁵cells/mL mixed cell suspension, 25 μL of which was added to a coatedcell culture container to seed the mixed cells.

Subsequently, cells were cultured for 2 hours in a cell incubator (37°C., 5% CO₂), and the medium was exchanged using a new medium. The cellswere then further cultured for 48 hours in a cell incubator (37° C., 5%CO₂) to obtain an aggregated cell structure.

Example VII-2

Except for mixing human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat at a ratio of 10 mesenchymalstem cells per 100 hepatoma cells, an aggregated cell structure wasobtained in a way similar to Example VII-1.

Example VII-3

Except for mixing human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat at a ratio of 50 mesenchymalstem cells per 100 hepatoma cells, an aggregated cell structure wasobtained in a way similar to Example VII-1.

Example VII-4

Except for mixing human hepatoma cells, adipose-derived mesenchymal stemcells of a GFP recombinant Lewis rat, and adhesive adipocytes derivedfrom Lewis rat adipose at a ratio of 33 mesenchymal stem cells and 33adhesive adipocytes per 100 hepatoma cells, an aggregated cell structurewas obtained in a way similar to Example VII-1.

Example VII-5

As the cells, human hepatoma cells (HepG2) stained in a similar way asin Example VII-1 were used as hepatocytes, adipose-derived mesenchymalstem cells (ADSC) of a GFP recombinant Lewis rat were used asfibroblasts, and macrophages were used as immune system cells. Themacrophages were used after inducing differentiation from monocytesderived from rat bone marrow (number BMM01, melomonocyte culturing kit,by Cosmo Bio).

As the first stage, human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat were mixed at a ratio of 25mesenchymal stem cells per 100 hepatoma cells, the mixed cells wereseeded in a way similar to Example VII-1, and after 5 hours, the cellshad adhered to form a single layer. The medium was then exchanged. Amacrophage suspension including macrophages was added onto the singlelayer to adjust the ratio to 5 macrophages per 100 human hepatoma cells,and static culturing was continued. Other than these differences,operations similar to Example VII-1 were performed.

When a single culture layer composed of hepatoma cells and mesenchymalstem cells aggregated like a drawstring bag, the macrophages that hadprecipitated onto the single culture layer were incorporated inside theaggregation, and one aggregated cell structure was obtained. Nomacrophages were confirmed on the exposed culturing surface, and it wasconfirmed that all of the seeded macrophages were incorporated in thecell aggregation.

Comparative Example VII-1

Except for mixing human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat at a ratio of 300 mesenchymalstem cells per 100 hepatoma cells, an aggregated cell structure wasobtained in a way similar to Example VII-1.

Comparative Example VII-2

Except for mixing human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat at a ratio of 100 mesenchymalstem cells per 100 hepatoma cells, an aggregated cell structure wasobtained in a way similar to Example VII-1.

Comparative Example VII-3

Except for producing a cell solution composed only of human hepatomacells, without mixing in adipose-derived mesenchymal stem cells of a GFPrecombinant Lewis rat, an aggregated cell structure was obtained in away similar to Example VII-1.

Comparative Example VII-4

Except for mixing human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat at a ratio of 5 mesenchymalstem cells per 100 hepatoma cells, an aggregated cell structure wasobtained in a way similar to Example VII-1.

Comparative Example VII-5

Except for mixing human hepatoma cells and adipose-derived mesenchymalstem cells of a GFP recombinant Lewis rat at a ratio of 67 mesenchymalstem cells per 100 hepatoma cells, an aggregated cell structure wasobtained in a way similar to Example VII-1.

[Evaluation]

(Appearance of Cell Structure)

The aggregated cell structures obtained in the Examples and ComparativeExamples (after 48 hours of culturing) were observed with a microscope(ECLIPSE-Ti by Nikon Corporation), and the appearance of the cells wasevaluated with the following criteria.

G (good): a spherical aggregated cell structure was obtained on all 8 ofthe coated culturing surfaces.P (poor): on one or more of the 8 coated culturing surfaces, the cellsthat adhered to the coated culturing surface remained as a single layerwithout aggregating, or a cell structure had a warped edge with acombination of an aggregated portion and an adhered portion not peeledoff from the coated culturing surface.

(Form of Cells in Cell Structure)

The seeded and cultured cells were observed with a microscope(ECLIPSE-Ti by Nikon Corporation) after being cultured for 2 hours, atwhich point the cells were adhered to the coated culturing surface. Theform of the hepatocytes among the adhered cells was evaluated with thefollowing criteria. The seeded hepatocytes (the hepatocytes beforeadhering to the coated culturing surface) had a cobblestone form in eachexample.

G (good): many hepatocytes were changed into a form similar tospindle-shaped fibroblasts.P (poor): no change in form of the hepatocytes was observed.

TABLE 6 Comparative Comparative Comparative Comparative ComparativeExample Example Example Example Example Example Example Example ExampleExample VII-1 VII-2 VII-3 VII-4 VII-5 VII-1 VII-2 VII-3 VII-4 VII-5Culture Cell Culture 35 mm 35 mm 35 mm 35 mm 35 mm 35 mm 35 mm 35 mm 35mm 35 mm Container Container dish dish dish dish dish dish dish dishdish dish Preparation Area of Culturing 900 900 900 900 900 900 900 900900 900 Step Surface of Cell Culture Container (mm²) Area of Each 3.143.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14 Coated Culturing Surface(mm²) Seeding Number of 33 10 50 33 25 300 100 0 5 67 Step Fibroblastsper 100 Hepatocytes Immune System no no no no yes no no no no no CellsAdded Number of — — — — — 5 — — — — Immune System Cells per 100Hepatocytes Adipocytes Added no no no yes no no no no no no Number of —— — 33 — — — — — — Adipocytes per 100 Hepatocytes Percentage of 75 91 6760 77 25 50 100 95 60 Hepatocytes among Total Number of Seeded Cells (%)Percentage of 25 9 33 20 19 75 50 0 5 40 Fibroblasts among Total Numberof Seeded Cells (%) Culturing Culturing 37° C. 37° C. 37° C. 37° C. 37°C. 37° C. 37° C. 37° C. 37° C. 37° C. Step Temperature Culturing Time 4848 48 48 48 48 48 48 48 48 (hours) Evaluation Appearance of G G G G G GG P P G Cell Structure Form of Cells in G G G G G P P P P P CellStructure

As is clear from Table 6, the hepatocytes changed from a cobblestoneform to a fibroblast-like form in the cell structures of the Examples inwhich hepatocytes and fibroblasts were mixed at a specific ratio.Therefore, the hepatocytes included in the cell structures of theExamples were highly invasive and transformed into highly malignantcancer cells. The cell structures of the Examples were thus judged to beuseable as a hepatic failure model. Upon crushing the cell structure ofeach Example, re-seeding the cells in a culture dish, and observing theform, the hepatocytes had the form of fibroblasts.

On the other hand, the hepatocytes in the cell structures of theComparative Examples remained in cobblestone form, with no observablechange. Upon crushing the cell structure of each Comparative Example,re-seeding the cells in a culture dish, and observing the form, thehepatocytes had a cobblestone form.

INDUSTRIAL APPLICABILITY

The present disclosure can provide a method of efficiently manufacturinga cell mass, a cell structure, or a three-dimensional tissue body.

In particular, Aspect (I) allows easy manufacturing of a chondrocytemass and a graft material that are useful for treatment of joints, thetrachea, the nose, and the like. Aspect (II) allows epithelial cells tobe cultured easily, allows a cell structure including epithelial cellsto be manufactured easily, and allows culturing of epithelial cells andthe manufacturing of cell structures thereof. Aspect (III) allows easyproduction of a three-dimensional tissue body with a ringed shape, aluminal shape, or the like and also allows easy production of athree-dimensional tissue body with a ringed shape, a luminal shape, orthe like. Aspect (IV) allows easy manufacturing of cell structureshaving a desired size and a well-defined spheroidal shape. Aspect (V)can control the aggregation mode of cells to manufacture cell structureswith a desired form. Aspect (VI) allows easy formation of a cellstructure that includes cardiomyocytes and fibroblasts and that isuseful as a heart disease model. Aspect (VII) allows easy formation of acell structure that includes hepatocytes and fibroblasts and that isuseful as a hepatic failure model.

1. A manufacturing method of a cell structure, the manufacturing methodcomprising: producing a coated region in which a culturing surface iscoated with a temperature-responsive polymer or a temperature-responsivepolymer composition, forming a droplet of a cell suspension in thecoated region, and performing cell culturing in the droplet; wherein asurface zeta potential of the coated region is 0 mV to 50 mV.
 2. Themanufacturing method of a cell structure of claim 1, wherein a contactangle of water relative to the coated region is 50° to 90°.
 3. Themanufacturing method of a cell structure of claim 1, comprisingproducing a plurality of the coated regions on the culturing surface. 4.The manufacturing method of a cell structure of claim 1, comprisingforming a plurality of the droplets on the coated region.
 5. Themanufacturing method of a cell structure of claim 1, wherein in eachcoated region, a bottom area of the droplet is smaller than an area ofthe coated region.
 6. The manufacturing method of a cell structure ofclaim 1, wherein the number of cells included in the droplet is 3.0×10⁵cells/mL or less.
 7. The manufacturing method of a cell structure ofclaim 1, wherein the droplet has a diameter of 1 μm to 8 mm.
 8. Themanufacturing method of a cell structure of claim 1, wherein an amountof the droplet is 0.5 μL to 50 μL.